Apparatus, systems, and methods for determining the concentration of microorganisms and the susceptibility of microorganisms to anti-infectives based on redox reactions

ABSTRACT

Various methods, devices, and systems for determining the concentration of microorganisms in a sample and determining the susceptibility of such microorganisms to one or more antibiotics or other types of anti-infectives are disclosed herein. More specifically, methods for quantifying microorganisms based on redox reactions are disclosed along with systems and devices for quantifying such microorganisms using certain oxidation reduction potential (ORP) sensors. Moreover, methods for determining the susceptibility and the degree of susceptibility of microorganisms to one or more anti-infectives are disclosed along with multiplex systems for such assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2018/054003 filed on Oct. 2, 2018, which claims the benefit ofU.S. Provisional Application No. 62/567,648 filed on Oct. 3, 2017, thecontents of which are incorporated herein by reference in theirentities.

TECHNICAL FIELD

The present disclosure relates generally to in vitro detection ofmicroorganisms or infectious agents and, more specifically, toapparatus, systems, and methods for determining the concentration ofmicroorganisms or infectious agents and the susceptibility of suchmicroorganisms or infectious agents to anti-infectives.

BACKGROUND

Infections caused by anti-infective resistant microorganisms orinfectious agents are a significant problem for healthcare professionalsin hospitals, nursing homes, and other healthcare environments. Rapiddetection of such microorganisms is crucial in order to prevent thespread of their resistance profiles. When faced with such an infection,a preferred course of action is for a clinician to use anti-infectivecompounds judiciously, preferably only those necessary to alleviate theinfection. However, what occurs most frequently today is that broadspectrum anti-infectives are given to the patient to ensure adequacy oftreatment. This tends to result in microorganisms with multipleanti-infective resistances. Ideally, the sensitivity of themicroorganism to anti-infectives would be detected soon after itspresence is identified.

Existing methods and instruments used to detect anti-infectiveresistance in microorganisms include costly and labor intensivemicrobial culturing techniques to isolate the microorganism and includetests such as agar disk diffusion or broth microdilution whereanti-infectives are introduced as liquid suspensions, paper disks, ordried gradients on agar media. However, those methods require manualinterpretation by skilled personnel and are prone to technical orclinician error.

While automated inspection of such panels or media can reduce thelikelihood of clinician error, current instruments used to conduct theseinspections are often complex and require the addition of reportermolecules or use of costly components such as transparent indium tinoxide (ITO) electrodes. In addition, current instruments often rely onan optical read-out of the investigated samples, which require bulkydetection equipment.

As a result of the above limitations and restrictions, there is a needfor improved apparatus, systems, and methods to quickly and effectivelydetect anti-infective resistant microorganisms in a patient sample.

SUMMARY

Various apparatus, systems and methods for detecting the susceptibilityof an infectious agent in a sample to one or more anti-infectives aredescribed herein. In one embodiment a method of determining aconcentration of an infectious agent can involve diluting a samplecomprising the infectious agent with a dilutive solution to yield adiluted sample. The method can further involve introducing the dilutedsample to a sensor such that the diluted sample is in fluidcommunication with a redox-active material of the sensor. The method canalso involve monitoring an oxidation reduction potential (ORP) of thediluted sample over a period of time using at least one parameteranalyzer coupled to the sensor to determine the concentration of theinfectious agent in the sample. The ORP can be monitored in the absenceof any added reporter molecules in the diluted sample.

In another embodiment, a system to determine a concentration of aninfectious agent is disclosed comprising a metering conduit configuredto deliver a dilutive solution to a sample comprising the infectiousagent to yield a diluted sample. The system can comprise a redox-activematerial, a sample delivery conduit configured to introduce the dilutedsample to the sensor, and at least one parameter analyzers coupled tothe sensor. The parameter analyzer can be configured to monitor an ORPof the diluted sample over a period of time when the diluted sample isin fluid communication with the redox-active material of the sensor. TheORP can be monitored in the absence of any added reporter molecules inthe diluted sample to determine the concentration of the infectiousagent in the sample.

In another embodiment, a method of determining a susceptibility of aninfectious agent to an anti-infective can involve diluting a samplecomprising the infectious agent with a dilutive solution to yield adiluted sample. The method can also involve separating the dilutedsample into a first aliquot and a second aliquot. The second aliquot canbe used as a control solution. The method can also involve mixing ananti-infective at a first concentration into the first aliquot to yielda test solution and introducing the test solution to a first sensor suchthat the test solution is in fluid communication with a redox-activematerial of the first sensor. The method can further involve introducingthe control solution to a second sensor such that the control solutionis in fluid communication with the redox-active material of the secondsensor. The method can also involve monitoring an ORP of the testsolution and the control solution over a period of time using one ormore parameter analyzers coupled to the first sensor, the second sensor,or a combination thereof. The ORPs can be monitored in the absence ofany added reporter molecules in the test solution or the controlsolution. The method can further involve comparing the ORP of the testsolution with the ORP of the control solution to determine thesusceptibility of the infectious agent to the anti-infective.

In yet another embodiment, a system to determine a susceptibility of aninfectious agent to one or more anti-infectives can comprise a meteringconduit configured to deliver a dilutive solution to a sample comprisingthe infectious agent to yield a diluted sample. The metering conduit canseparate the diluted sample into a first aliquot and a second aliquot.The second aliquot can be used as a control solution. The system canalso comprise a first sensor comprising a redox-active material and asecond sensor comprising the redox-active material.

The system can also comprise a first sample delivery conduit configuredto introduce the first aliquot to the first sensor. The first sampledelivery conduit can comprise a first anti-infective at a firstconcentration. The first aliquot can mix with the first anti-infectiveto form a first test solution. The system can also comprise a secondsample delivery conduit configured to introduce the control solution tothe second sensor.

The system can further comprise one or more parameter analyzers coupledto the first sensor and the second sensor. The one or more parameteranalyzers can monitor an ORP of the first test solution over a period oftime when the first test solution is in fluid communication with theredox-active material of the first sensor. The ORP can be monitored inthe absence of any added reporter molecules in the first test solution.The one or more parameter analyzers can also monitor the ORP of thecontrol solution over a period of time when the control solution is influid communication with the redox-active material of the second sensor.The ORP can be monitored in the absence of any added reporter moleculesin the control solution. The one or more parameter analyzers or anotherdevice within the system can compare the ORP of the first test solutionwith the ORP of the control solution to determine the susceptibility ofthe infectious agent to the first anti-infective.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one embodiment of a method for determining theconcentration of one or more infectious agents in a biological sample.

FIGS. 2A to 2C illustrate embodiments of systems for determining theconcentration of one or more infectious agents in a biological sample.

FIG. 3A illustrates example growth curves used to generate a standardcurve for determining the concentration of one or more infectious agentsin a biological sample.

FIG. 3B illustrates a fitted standard curve for determining theconcentration of one or more infectious agents in a biological sample.

FIG. 4 illustrates example bacterial growth curves used to determine theconcentration of the bacteria in a sample.

FIG. 5 illustrates one embodiment of a method for determining thesusceptibility of one or more infectious agents to one or moreanti-infectives.

FIG. 6 illustrates one embodiment of a multiplex system for determiningthe susceptibility of one or more infectious agents to one or moreanti-infectives.

FIG. 7A illustrates a growth curve of an infectious agent resistant toone or more anti-infectives.

FIG. 7B illustrates a growth curve of an infectious agent susceptible toone or more anti-infectives.

FIG. 8 illustrates growth curves of bacteria in the presence of certainanti-infectives.

FIG. 9A illustrates a schematic of an embodiment of a sensor used aspart of the methods and systems described herein.

FIG. 9B illustrates a schematic of another embodiment of the sensor usedas part of the methods and systems described herein.

DETAILED DESCRIPTION

Variations of the devices, systems, and methods described herein arebest understood from the detailed description when read in conjunctionwith the accompanying drawings. It is emphasized that, according tocommon practice, the various features of the drawings may not be toscale. On the contrary, the dimensions of the various features may bearbitrarily expanded or reduced for clarity and not all features may bevisible or labeled in every drawing. The drawings are taken forillustrative purposes only and are not intended to define or limit thescope of the claims to that which is shown.

FIG. 1 illustrates an embodiment of a method 100 for determining theconcentration of one or more infectious agents 102 in a sample 104. Themethod 100 can comprise introducing one or more aliquots of the sample104 into one or more reaction vessels 106 in step 1A. The reactionvessels 106 can refer to one or more test tubes, reaction tubes, wellsof a high throughput assay plate or well plate such as a 96-well plate,a 192-well plate, or a 384-well plate, culture plates or dishes, orother suitable containers for housing biological samples. One or morefluid delivery conduits 108 can inject, deliver, or otherwise introducethe aliquots of the sample 104 to the one or more reaction vessels 106.

In other embodiments not shown in FIG. 1, a stimulus solution can beadded to the sample 104 before introducing the sample 104 to thereaction vessel 106. The stimulus solution can be a nutrient or growthsolution. In these and other embodiments, the sample 104 can also befiltered before step 1A. This filtering step can involve filtering thesample 104 using an instance of a filter, a microfluidic filter, or acombination thereof to filter out debris, inorganic material, and largercellular components including blood cells or epithelial cells from thesample 104.

The sample 104 can comprise at least one of a biological sample, abodily fluid, a wound swab or sample, a rectal swab or sample, and abacterial culture derived from the biological sample, the bodily fluid,the wound swab or sample, or the rectal swab or sample. The bodily fluidcan comprise urine, blood, serum, plasma, saliva, sputum, semen, breastmilk, joint fluid, spinal fluid, wound material, mucus, fluidaccompanying stool, re-suspended rectal or wound swabs, vaginalsecretions, cerebrospinal fluid, synovial fluid, pleural fluid,peritoneal fluid, pericardial fluid, amniotic fluid, cultures of bodilywhich has been tested positive for bacteria or bacterial growth such asblood culture which has been tested positive for bacteria or bacterialgrowth (i.e., positive blood culture), or a combination thereof.

The infectious agents 102 that can be quantified using the methods orsystems disclosed herein can be any metabolizing single- ormulti-cellular organism including bacteria and fungi. In certainembodiments, the infectious agent 102 can be bacteria selected from thegenera Acinetobacter, Acetobacter, Actinomyces, Aerococcus, Aeromonas,Agrobacterium, Anaplasma, Azorhizobium, Azotobacter, Bacillus,Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia,Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila,Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia,Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium,Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus,Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus,Morganella, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Pandoraea,Pasteurella, Peptostreptococcus, Porphyromonas, Prevotella, Proteus,Providencia, Pseudomonas, Ralstonia, Raoultella, Rhizobium, Rickettsia,Rochalimaea, Rothia, Salmonella, Serratia, Shewanella, Shigella,Spirillum, Staphylococcus, Strenotrophomonas, Streptococcus,Streptomyces, Treponema, Vibrio, Wolbachia, Yersinia, or a combinationthereof. In other embodiments, the infectious agent 102 can be one ormore fungi selected from the genera Candida or Cryptococcus or mold.

Other specific bacteria that can be quantified using the methods andsystems disclosed herein can comprise Staphylococcus aureus,Staphylococcus lugdunensis, coagulase-negative Staphylococcus species(including but not limited to Staphylococcus epidermidis, Staphylococcushaemolyticus, Staphylococcus hominis, Staphylococcus capitis, notdifferentiated), Enterococcus faecalis, Enterococcus faecium (includingbut not limited to Enterococcus faecium and other Enterococcus spp., notdifferentiated, excluding Enterococcus faecalis), Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,Streptococcus spp., (including but not limited to Streptococcus mitis,Streptococcus pyogenes, Streptococcus gallolyticus, Streptococcusagalactiae, Streptococcus pneumoniae, not differentiated), Pseudomonasaeruginosa, Acinetobacter baumannii, Klebsiella spp. (including but notlimited to Klebsiella pneumoniae, Klebsiella oxytoca, notdifferentiated), Escherichia coli, Enterobacter spp. (including but notlimited to Enterobacter cloacae, Enterobacter aerogenes, notdifferentiated), Proteus spp. (including but not limited to Proteusmirabilis, Proteus vulgaris, not differentiated), Citrobacter spp.(including but not limited to Citrobacter freundii, Citrobacter koseri,not differentiated), Serratia marcescens, Candida albicans, and Candidaglabrata.

Other more specific bacteria that can be quantified can compriseAcinetobacter baumannii, Actinobacillus spp., Actinomycetes, Actinomycesspp. (including but not limited to Actinomyces israelii and Actinomycesnaeslundii), Aeromonas spp. (including but not limited to Aeromonashydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), andAeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans,Actinobacillus actinomycetemcomitans, Bacillus spp. (including but notlimited to Bacillus anthracis, Bacillus cereus, Bacillus subtilis,Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroidesspp. (including but not limited to Bacteroides fragilis), Bartonellaspp. (including but not limited to Bartonella bacilliformis andBartonella henselae, Bifidobacterium spp., Bordetella spp. (includingbut not limited to Bordetella pertussis, Bordetella parapertussis, andBordetella bronchiseptica), Borrelia spp. (including but not limited toBorrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (includingbut not limited to Brucella abortus, Brucella canis, Brucellamelintensis and Brucella suis), Burkholderia spp. (including but notlimited to Burkholderia pseudomallei and Burkholderia cepacia),Campylobacter spp. (including but not limited to Campylobacter jejuni,Campylobacter coli, Campylobacter lari and Campylobacter fetus),Capnocytophaga spp., Cardiobacterium hominis, Chlamydia trachomatis,Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter spp.Coxiella burnetii, Corynebacterium spp. (including but not limited to,Corynebacterium diphtheriae, Corynebacterium jeikeum andCorynebacterium), Clostridium spp. (including but not limited toClostridium perfringens, Clostridium difficile, Clostridium botulinumand Clostridium tetani), Eikenella corrodens, Enterobacter spp.(including but not limited to Enterobacter aerogenes, Enterobacteragglomerans, Enterobacter cloacae and Escherichia coli, includingopportunistic Escherichia coli, including but not limited toenterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E.coli, enterohemorrhagic E. coli, enteroaggregative E. coli anduropathogenic E. coli) Enterococcus spp. (including but not limited toEnterococcus faecalis and Enterococcus faecium) Ehrlichia spp.(including but not limited to Ehrlichia chafeensia and Ehrlichia canis),Erysipelothrix rhusiopathiae, Eubacterium spp., Francisella tularensis,Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum,Haemophilus spp. (including but not limited to Haemophilus influenzae,Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae,Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacterspp. (including but not limited to Helicobacter pylori, Helicobactercinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella spp.(including but not limited to Klebsiella pneumoniae, Klebsiellagranulomatis and Klebsiella oxytoca), Lactobacillus spp., Listeriamonocytogenes, Leptospira interrogans, Legionella pneumophila,Leptospira interrogans, Peptostreptococcus spp., Moraxella catarrhalis,Morganella spp., Mobiluncus spp., Micrococcus spp., Mycobacterium spp.(including but not limited to Mycobacterium leprae, Mycobacteriumtuberculosis, Mycobacterium intracellulare, Mycobacterium avium,Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm spp.(including but not limited to Mycoplasma pneumoniae, Mycoplasma hominis,and Mycoplasma genitalium), Nocardia spp. (including but not limited toNocardia asteroides, Nocardia cyriacigeorgica and Nocardiabrasiliensis), Neisseria spp. (including but not limited to Neisseriagonorrhoeae and Neisseria meningitidis), Pasteurella multocida,Plesiomonas shigelloides. Prevotella spp., Porphyromonas spp.,Prevotella melaninogenica, Proteus spp. (including but not limited toProteus vulgaris and Proteus mirabilis), Providencia spp. (including butnot limited to Providencia alcalifaciens, Providencia rettgeri andProvidencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes,Rhodococcus equi, Rickettsia spp. (including but not limited toRickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii,Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) andRickettsia typhi), Rhodococcus spp., Serratia marcescens,Stenotrophomonas maltophilia, Salmonella spp. (including but not limitedto Salmonella enterica, Salmonella typhi, Salmonella paratyphi,Salmonella enteritidis, Salmonella cholerasuis and Salmonellatyphimurium), Serratia spp. (including but not limited to Serratiamarcesans and Serratia liquifaciens), Shigella spp. (including but notlimited to Shigella dysenteriae, Shigella flexneri, Shigella boydii andShigella sonnei), Staphylococcus spp. (including but not limited toStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcushemolyticus, Staphylococcus saprophyticus), Streptococcus spp.(including but not limited to Streptococcus pneumoniae (for examplechloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,erythromycin-resistant serotype 14 Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema spp. (includingbut not limited to Treponema carateum, Treponema petenue, Treponemapallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasmaurealyticum, Veillonella sp., Vibrio spp. (including but not limited toVibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrioparahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibriomimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibriodamsela and Vibrio furnisii), Yersinia spp. (including but not limitedto Yersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis) and Xanthomonas maltophilia among others.

Furthermore, other infectious agents 102 that can be quantified cancomprise fungi or mold including, but not limited to, Candida spp.(including but not limited to Candida albicans, Candida glabrata,Candida tropicalis, Candida parapsilosis, and Candida krusei),Aspergillus spp. (including but not limited to Aspergillus fumigatous,Aspergillus flavus, Aspergillus clavatus), Cryptococcous spp. (includingbut not limited to Cryptococcus neoformans, Cryptococcus gattii,Cryptococcus laurentii, and Cryptococcus albidus), Fusarium spp.(including but not limited to Fusarium oxysporum, Fusarium solani,Fusarium verticillioides, and Fusarium proliferatum), Rhizopus oryzae,Penicillium marneffei, Coccidiodes immitis, and Blastomycesdermatitidis.

The fluid delivery conduits 108 can include tubes, pumps, containers, ormicrofluidic channels for delivering buffers, reagents, fluid samplesincluding the sample 104 or solubilized solutions thereof, othersolutions, or a combination thereof to and between devices, apparatus,or containers in the system. For example, as shown in FIG. 1, the fluiddelivery conduits 108 can refer to parts of a pump such as a syringepump. In other embodiments, the fluid delivery conduits 108 can includeor refer to at least part of a hydraulic pump, a pneumatic pump, aperistaltic pump, a vacuum pump or a positive pressure pump, a manual ormechanical pump, or a combination thereof. In additional embodiments,the fluid delivery conduits 108 can include or refer to at least part ofan injection cartridge, a pipette, a capillary, or a combinationthereof. The fluid delivery conduits 108 can also be part of a vacuumsystem configured to draw fluid to or through channels, tubes, orpassageways under vacuum. Moreover, the fluid delivery conduits 108 caninclude or refer to at least part of a multichannel delivery system orpipette.

The method 100 can comprise diluting the sample 104 comprising theinfectious agent 102 with a dilutive solution 110 to yield a dilutedsample 112 in step 1B. In one embodiment, the dilutive solution 110 cancomprise growth media or a growth inducer. In this and otherembodiments, the dilutive solution 110 can be a solution containingbacto-tryptone, yeast extract, beef extract, cation-adjusted MuellerHinton Broth (CAMHB), Mueller Hinton growth media (MHG), starch, acidhydrolysate of casein, calcium chloride, magnesium chloride, sodiumchloride, blood or lysed blood including lysed horse blood (LHB),CAMHB-LHB, glucose, or a combination thereof. The growth inducer cancomprise a carbon-based inducer, a nitrogen-based inducer, a mineral, atrace element, a biological growth factor, or any combination thereof.For example, the growth inducer can include but is not limited toglucose, ammonia, magnesium, blood, or a combination thereof. In oneexample embodiment, the dilutive solution 110 can comprise Tryptone,yeast extract, sodium chloride, and glucose. The dilutive solution 110can be used to counteract the buffering effects of ions or substancespresent in the sample 104.

In one embodiment, diluting the sample 104 with the dilutive solution110 in step 1B can involve diluting the sample 104 to a dilution ratiobetween about 1:1 to about 1:10. In another embodiment, diluting thesample 104 with the dilutive solution 110 can involve diluting thesample 104 to a dilution ratio between about 1:10 to about 1:100. In yetanother embodiment, diluting the sample 104 with the dilutive solution110 can involve diluting the sample 104 to a dilution ratio betweenabout 1:100 to about 1:1000. In a further embodiment, diluting thesample 104 with the dilutive solution 110 can involve diluting thesample 104 to a dilution ratio between about 1:1000 to about 1:10000.Although FIG. 1 illustrates one reaction vessel 106 or one aliquot ofthe sample 104 being diluted, it is contemplated by this disclosure thatmultiple aliquots of the sample 104 can be diluted to different dilutionratios such that one or more diluted samples 112 can act as internalcontrols.

As will be discussed in the following sections in relation to FIGS. 2A,2B, and 2C, in alternative embodiments, the method 100 can comprisediluting the sample 104 comprising the infectious agent 102 withdeionized water, a saline solution, or a combination thereof serving asthe dilutive solution 110. In these embodiments, the diluted sample(s)112 can be introduced to one or more sensors through sample deliveryconduits comprising growth media or a growth inducer such that thediluted sample 112 is mixed with the growth media or growth inducer.More details concerning these embodiments will be discussed in thefollowing sections.

The method 100 can also comprise incubating the diluted sample 112 at anelevated temperature for a period of time in step 1C. The diluted sample112 can be incubated in the same reaction vessel 106 or transferred to adifferent reaction vessel 106 or container. For example, the dilutedsample 112 can be heated to a temperature of between about 30° C. andabout 40° C. (e.g., 35° C.±2° C.) and allowed to incubate for anincubation period 114. The incubation period 114 can range from 15minutes to over one hour. In other embodiments, the incubation period114 can be less than 15 minutes or up to 48 hours.

The method 100 can further comprise introducing the diluted sample 112to a sensor 116 or exposing the sensor 116 to the diluted sample 112such that the diluted sample 112 is in fluid communication with aredox-active material 908 (see FIGS. 9A and 9B) of the sensor 116 instep 1D. In one or more embodiments, the sensor 116 can be an oxidationreduction potential (ORP) sensor configured to respond to a change in asolution characteristic (e.g., the ORP) of a measured solution. In theexample embodiment shown in FIG. 1, exposing the sensor 116 to thediluted sample 112 can involve directly immersing at least part of ahandheld or probe instance of the sensor 116 into the diluted sample112. In this embodiment, the handheld or probe instance of the sensor116 can be a handheld OPR sensor coupled to a standalone parameteranalyzer 118 such as a voltmeter or multimeter. In another exampleembodiment shown in FIG. 2, introducing the diluted sample 112 to thesensor 116 can involve injecting, delivering, or otherwise introducingthe diluted sample 112 to a well or container comprising the sensor 116fabricated on a substrate. The sensor 116 will be discussed in moredetail in the following sections.

The method 100 can further comprise monitoring the ORP of the dilutedsample 112 with at least one parameter analyzer 118 coupled to thesensor 116 in step 1E. The ORP of the diluted sample 112 can bemonitored in the absence of any added reporter molecules or exogenousreporter molecules in the diluted sample 112 in order to determine theconcentration of the infectious agent 102 in the original sample 104.

The diluted sample 112 can have a solution characteristic. The solutioncharacteristic of the diluted sample 112 can change as the amount ofelectro-active redox species changes due to the energy use, oxygenuptake or release, growth, or metabolism of the infectious agents 102 inthe diluted sample 112. For example, the amount of electro-active redoxspecies in the diluted sample 112 can change as a result of cellularactivity (e.g., microbial aerobic or anaerobic respiration) undertakenby the infectious agents 102. As a more specific example, the amount ofelectron donors from Table 1 below (e.g., the amount of energy carrierssuch as nicotinamide adenine dinucleotide (NADH) and flavin adeninedinucleotide (FADH₂)) in the diluted sample 112 can change due to thegrowth of the infectious agents 102 in the diluted sample 112 within thereaction vessel 106. Also, as another more specific example, the amountof oxygen depleted in the diluted sample 112 due to aerobic respirationcan change due to the growth of the infectious agents 102 in the dilutedsample 112 within the reaction vessel 106.

TABLE 1 Below is a “redox tower” visualizing potential electron donorsand acceptors which can be utilized by microorganisms or infectiousagents during the course of metabolism. An electron donor will have agreater negative potential than the electron acceptor. In aerobicrespiration for example, O₂ can serve as a terminal electron acceptorwhereas in anaerobic respiration, the terminal electron acceptor cancomprise NO₃ ⁻, Fe³⁺, Mn⁴⁺, SO₄ ²⁻, or CO₂. Measured Standard StandardReduction Reduction Potential E′₀ Potential E′₀ Electron Donor andAcceptor Pairs (mV) (mV) range Glucose

 2 Pyruvate + 2e⁻ −720 −700 −600 Glucose

 6 CO₂ + 24e⁻ −500 −500 H₂

 2H⁺ + 2e⁻ −420 −400 NADH

 NAD⁺ + 2e⁻ −320 −300 2 GSH

 GSSG + 2e⁻ −240 −200 H₂S

 SO₄ ²⁻ + 8e⁻ −220 FADH₂

 FAD + 2H⁺ + 2e⁻ −220 Lactate

 Pyruvate + 2e⁻ −190 −100 Succinate

 Fumarate + 2e⁻ 33 0 Cyt b (red)

 Cyt b (ox) + e⁻ 80 Ubiquinol

 Ubiquinone + 2e⁻ 110 100 Cyt c (red)

 Cyt c (ox) + e⁻ 250 200 Cyt a (red)

 Cyt a (ox) + e⁻ 290 300 NO₂ ⁻ + H₂O

 NO₃ ⁻ + 2e⁻ 420 400 NH₄ ⁺ + H₂O

 NO₂ ⁻ + 6e⁻ 440 Mn²⁺ + H₂O

 MnO₂ + 2e⁻ 460 500 600 ½ N₂ + 3H₂O

 NO₃ ⁻ + 5e⁻ 740 700 Fe²⁺

 Fe³⁺ + 1e⁻ 770 H₂O

 ½ O₂ + 2H⁺ + 2e⁻ 820 800 900

As illustrated in FIG. 1, the parameter analyzer 118 can be connected toor communicatively coupled to a device having a display 122 or a displaycomponent configured to display a read-out of the electricalcharacteristic of the sensor 116 representing the solutioncharacteristic of the diluted sample 112. Such a device can be referredto as a reader 120. In certain embodiments, the reader 120 can be amobile device, a handheld device, a tablet device, or a computing devicesuch as a laptop or desktop computer and the display 122 can be a mobiledevice display, a handheld device display, a tablet display, or a laptopor desktop monitor. In these and other embodiments, the parameteranalyzer 118 can wirelessly communicate a signal or result to the reader120 or another computing device having the display 122. In otherembodiments, the parameter analyzer 118 and the reader 120 can beintegrated into one device.

The method 100 can further comprise monitoring the ORP of the dilutedsample 112 over a period of time with the at least one parameteranalyzer 118, the reader 120, or a combination thereof in step 1F. Theparameter analyzer 118, the reader 120, or a combination thereof canalso determine the concentration of the infectious agent 102 in thesample 104 within this period of time in step 1F. The period of timewithin which the parameter analyzer 118, the reader 120, or acombination thereof can determine the concentration of the infectiousagent 102 can be referred to as a quantification window 124. In oneembodiment, the quantification window 124 can be between 60 minutes and120 minutes. In other embodiments, the quantification window 124 can bebetween 5 minutes and 60 minutes. In additional embodiments, thequantification window 124 can be greater than 120 minutes.

The parameter analyzer, the reader 120, or a combination thereof candetermine the concentration of the infectious agent 102 in the sample104 using measured ORP signals (e.g., measured output voltages) and astandard curve 126 generated by monitoring the ORPs of prepared culturesof the infectious agent in different concentrations. In someembodiments, the standard curve 126 can be generated before step 1A. Inother embodiments, the standard curve 126 can be generated at any timeprior to step 1F.

In one example embodiment, the standard curve 126 can be generated usingdifferent concentrations of bacteria (e.g., from about 1*10⁴ CFU/mL toabout 1*10⁸ CFU/mL) grown at 35° C. in growth media. The ORPs of growthmedia comprising such bacterial concentrations can be monitored overtime for a change in their ORPs using an ORP sensor. A threshold voltagecan be set (e.g., between about −100 mV and 100 mV) and a standard curvecan be generated by plotting the various bacterial concentrationsagainst the time it took the monitored ORP of each such bacterialconcentration to reach the threshold voltage (also known as thetime-to-detection (TTD)). Generation of the standard curve is discussedin more detail in the following sections.

With the standard curve 126 generated, the method 100 can involvecomparing the measured or monitored ORP of the diluted sample 112 overtime against the values obtained from the standard curve 126. Forexample, as shown in FIG. 1, a growth curve 128 for the infectious agent102 within the sample 104 under investigation can be generated using thechange in ORP of the diluted sample 112 over time measured by theparameter analyzer 118, the reader 120, or a combination thereof. Thesame threshold voltage 130 can be applied to the growth curve 128 as thethreshold voltage 130 used to generate the standard curve 126. Thetime-to-detection 132 or the time it took the monitored ORP of thediluted sample 112 to reach the threshold voltage 130 can be ascertainedfrom the growth curve 128. The reader 120, the parameter analyzer 118,or another device can then determine the concentration of the infectiousagent in the sample 104 under investigation by using thetime-to-detection 132 and the values obtained from the standard curve126. For example, the concentration can be calculated using thetime-to-detection 132 and an equation derived from the standard curve126.

In some embodiments, one or more of the aforementioned steps of themethod 100 can be stored as machine-executable instructions or logicalcommands in a non-transitory machine-readable medium (e.g., a memory orstorage unit) of the parameter analyzer 118, the reader 120, or anotherdevice communicatively or electrically coupled to the parameter analyzer118 or the reader 120. Any of the parameter analyzer 118, the reader120, or another device coupled to the parameter analyzer 118 or thereader 120 can comprise one or more processors or controllers configuredto execute the aforementioned instructions or logical commands.

The steps depicted in FIG. 1 do not require the particular order shownto achieve the desired result. Moreover, certain steps or processes maybe omitted or occur in parallel in order to achieve the desired result.In addition, any of the systems or devices disclosed herein can be usedin lieu of devices or systems shown in the steps of FIG. 1.

FIGS. 2A, 2B, and 2C illustrate embodiments of systems 200 fordetermining the concentration of one or more infectious agents 102 in asample 104 (see FIG. 1). It is contemplated by this disclosure (and itshould be understood by one or ordinary skill in the art) that any ofthe systems 200 described in connection with FIG. 2A, 2B, or 2C can beused to undertake one or more steps of the method 100 described in thepreceding sections. FIG. 2A illustrates that the system 200 can compriseone or more sensors 116 fabricated or positioned on a surface of asubstrate 202, one or more parameter analyzers 118 electrically orcommunicatively coupled to the one or more sensors 116, and one or morereaders 120 electrically or communicatively coupled to the one or moreparameter analyzers 118. In some embodiments, the reader 120 and theparameter analyzer 118 can be integrated into one device.

In some embodiments, the substrate 202 and the sensors 116 can be partof a cartridge, a test strip, an integrated circuit, amicro-electro-mechanical system (MEMS) device, a microfluidic chip, or acombination thereof. In these and other embodiments, the substrate 202can be part of a lab-on-a-chip (LOC) device. In all such embodiments,the sensors 116 can comprise components of such circuits, chips, ordevices including, but not limited to, one or more transistors, gates,or other electrical components. The sensors 116 can be micro- ornano-scale ORP sensors. Each of the sensors 116 can comprise an activeelectrode and a reference electrode (see FIGS. 9A and 9B). Each of thesensors 116 can also comprise a redox-active material 908 (see FIGS. 9Aand 9B) or layer such as a gold layer, a platinum layer, a metal oxidelayer, carbon layer, or a combination thereof. The sensors 116 will bediscussed in more detail in the following sections.

In one embodiment, the sample 104 comprising the infectious agent 102can be diluted using growth media or growth inducers representing thedilutive solution 110. The growth media or growth inducers can be thesame growth media or growth inducers described with respect to step 1Bof method 100. In this embodiment, the diluted sample 112 can beinjected, pipetted, delivered, or otherwise introduced to the one ormore sensors 116 such that the diluted sample 112 is in fluidcommunication with the redox-active material 908 (see FIGS. 9A and 9B)of the sensors 116.

The system 200 can also comprise an incubating component configured toincubate the diluted sample 112 in fluid communication with the sensor116 by heating the diluted sample 112 to a temperature of between about30° C. and about 40° C. (e.g., 35° C.±2° C.) for a period of time (e.g.,the incubation period 114).

In another embodiment, the sample 104 comprising the infectious agent102 can be diluted using deionized water, a saline solution, or acombination thereof representing the dilutive solution 110 to yield thediluted sample 112. In this embodiment, the one or more sensors 116 onthe substrate 202 can be covered or coated by a lyophilized or driedform of the growth media or growth inducer. For example, the one or moresensors 116 can comprise a layer of lyophilized or dried growth media orgrowth inducer covering or coating the one or more sensors 116. Inanother embodiment, the lyophilized or dried growth inducer can cover orcoat a surface in a vicinity of the one or more sensors 116. In yetanother embodiment, the one or more sensors 116 can be disposed within awell or a container defined on the substrate 202 and the well orcontainer can comprise an aqueous form of the growth media or growthinducer. In all such embodiments, the diluted sample 112 can mix withthe growth media or growth inducer.

The incubating component can then incubate the diluted sample 112 mixedwith the growth media or growth inducer by heating the mixture to atemperature of between about 30° C. and about 40° C. (e.g., 35° C.±2°C.) for a period of time (e.g., the incubation period 114).

FIG. 2B illustrates another embodiment of a system 200 for determiningthe concentration of one or more infectious agents 102 in a sample 104.The system 200 can comprise a sample receiving surface 204 defined on asubstrate 202, one or more metering conduits 206 in fluid communicationwith the sample receiving surface 204, a sensor 116 fabricated orotherwise disposed on the substrate 202, one or more sample deliveryconduits 208 fluidly connecting or extending in between the samplereceiving surface 204 and the sensor 116, a parameter analyzer 118electrically or communicatively coupled to the sensor 116, and a reader120 electrically or communicatively coupled to the parameter analyzer118. In some embodiments, the reader 120 and the parameter analyzer 118can be integrated into one device.

In one or more embodiments, the sample receiving surface 204 can be aflat surface for receiving the sample 104. In other embodiments, thesample receiving surface 204 can be a concave or tapered surface of awell, divot, dish, or container. For example, the sample 104 can beinjected, pipetted, pumped, spotted, or otherwise introduced to thesample receiving surface 204.

The one or more metering conduits 206 can be channels, passageways,capillaries, tubes, parts therein, or combinations thereof fordelivering the dilutive solution 110 to the sample 104 on the samplereceiving surface 204. For example, the one or more metering conduits206 can refer to channels, passageways, capillaries, or tubes defined onthe substrate 202. Also, for example, the one or more metering conduits206 can refer to channels, passageways, capillaries, or tubes serving aspart of hydraulic pump, a pneumatic pump, peristaltic pump, a vacuum orpositive pressure pump, a manual or mechanical pump, a syringe pump, ora combination thereof. For example, the one or more metering conduits206 can be microfluidic channels or tubes or channels serving as part ofa vacuum system.

In some embodiments, the one or more metering conduits 206 can beconfigured to dilute the sample 104 with the dilutive solution 110 to adilution ratio between about 1:1 to about 1:10. In other embodiments,the one or more metering conduits 206 can be configured to dilute thesample 104 with the dilutive solution 110 to a dilution ratio betweenabout 1:10 to about 1:100. In additional embodiments, the one or moremetering conduits 206 can be configured to dilute the sample 104 withthe dilutive solution 110 to a dilution ratio between about 1:100 toabout 1:1000. In yet additional embodiments, the one or more meteringconduits 206 can be configured to dilute the sample 104 with thedilutive solution 110 to a dilution ratio between about 1:1000 to about1:10000.

The one or more sample delivery conduits 208 can be channels,passageways, capillaries, tubes, parts therein, or combinations thereoffor delivering the diluted sample 112 to the sensor 116. For example,the one or more sample delivery conduits 208 can fluidly connect thesample receiving surface 204 with the sensor 116 such that the dilutedsample 112 or fluid on the sample receiving surface 204 is in fluidcommunication with at least part of the sensor 116.

As shown in the example embodiment of FIG. 2B, the one or more sampledelivery conduits 208 can comprise growth media 210 or growth inducer.The growth media 210 or growth inducer can be the same growth media orgrowth inducer discussed in connection with FIG. 2A and FIG. 1.

In one or more embodiments, the sample delivery conduits 208 can becovered or coated by a lyophilized or dried form of the growth media 210or the growth inducer. In other embodiments, the sample deliveryconduits 208 can contain growth media 210 or grow inducer in an aqueousform. In these and other embodiments, the dilutive solution 110delivered by the one or more metering conduits 206 can be a salinesolution, deionized water, or a combination thereof. The dilutivesolution 110 can dilute the sample 104 and deliver the sample 104through the sample delivery conduits 208 to the sensor 116 such that thediluted sample 112 mixes with the growth media 210 en route to thesensor 116. In other embodiments not shown in the figures, at least onelayer of the sensor 116 or a surface in a vicinity of the sensor 116 canbe coated or covered by the growth media 210 in lyophilized or driedform and the diluted sample 112 can mix with the growth media 210 whenthe diluted sample 112 is in fluid communication with the part of thesensor 116 or part of the area covered by the growth media 210.

In all such embodiments, the diluted sample 112 can mix with the growthmedia 210 or growth inducer.

The incubating component can then incubate the diluted sample 112 mixedwith the growth media 210 or growth inducer by heating the mixture to atemperature of between about 30° C. and about 40° C. (e.g., 35° C.±2°C.) for a period of time (e.g., the incubation period 114).

In some embodiments, the substrate 202 and sensors 116 can be part of acartridge, a test strip, an integrated circuit, amicro-electro-mechanical system (MEMS) device, a microfluidic chip, or acombination thereof. In these and other embodiments, the substrate 202can be part of a lab-on-a-chip (LOC) device. In all such embodiments,the sensor 116 can comprise components of such circuits, chips, ordevices including, but not limited to, one or more transistors, gates,or other electrical components. The sensor 116 can be a micro- ornano-scale ORP sensor. The sensor 116 can comprise an active electrodeand a reference electrode. The sensor 116 can also comprise aredox-active material 908 (see FIGS. 9A and 9B) or layer such as a goldlayer, a platinum layer, a metal oxide layer, carbon layer, or acombination thereof. The sensor 116 will be discussed in more detail inthe following sections.

FIG. 2C illustrates a multiplex version of the system 200 shown in FIG.2B. For example, the system 200 of FIG. 2C can have multiple sensors116, multiple metering conduits 206, and multiple sample deliveryconduits 208. In one embodiment, different samples comprising differenttypes of infectious agents can be delivered, injected, or otherwiseintroduced to the various sample receiving surfaces 204 on one substrate202.

The substrate 202 can be comprised of a polymeric material, a metal, aceramic, a semiconductor layer, an oxide layer, an insulator, or acombination thereof. The substrate 202 can be part of a test strip,cartridge, chip or lab-on-a-chip, microfluidic device, multi-wellcontainer, or a combination thereof. The sensors 116 can be fabricatedor located on a surface of the substrate 202. In some embodiments, theone or more parameter analyzers 118 can also be fabricated or located onthe substrate 202. In other embodiments, the one or more parameteranalyzers 118 can be standalone devices such as a voltmeter or amultimeter electrically coupled to the sensors 116.

In this embodiment, the system 200 shown in FIG. 2C can be used todetermine the concentrations of infectious agents 102 in multiplesamples concurrently. In other embodiments, aliquots of the same sample104 can be introduced to the various sample receiving surfaces 204 onone substrate 202 and different amounts of the dilutive solution 110 canbe delivered to the various sample receiving surfaces 204 through themetering conduits 206. In this embodiment, the multiplex system 200 ofFIG. 2C can be used to dilute aliquots of the same sample 104 todifferent dilution ratios so as to use certain dilutions as internalcontrols and to determine the minimum amount of dilution needed toquantify a certain sample.

In the example embodiments shown in FIGS. 2A, 2B, and 2C, the one ormore parameter analyzers 118 can be disposed or fabricated on thesubstrate 202 or the parameter analyzers 118 can also be standalonedevices coupled to the one or more sensors 116. The parameter analyzers118 can be electrically or communicatively coupled to one or morereaders 120 having a display 122 or display component. The display 122or display component can be configured to display a read-out of theelectrical characteristic of the one or more sensors 116 representingthe solution characteristic of the diluted sample 112. In certainembodiments, the reader 120 can be a mobile device, a handheld device, atablet device, or a computing device such as a laptop or desktopcomputer and the display 122 can be a mobile device display, a handhelddevice display, a tablet display, or a laptop or desktop monitor. Insome embodiments, the parameter analyzer 118 can wirelessly communicatea signal or result to the reader 120 or another computing device havingthe display 122.

Similar to step 1F of method 100, the systems 200 of FIGS. 2A, 2B, and2C can monitor the ORP of the diluted sample 112 and determine theconcentration of the infectious agent 102 in the sample 104 within aperiod of time (e.g., the quantification window 124 of method 100). Thisperiod of time can be between 60 minutes and 120 minutes. In otherembodiments, this period of time can be between 5 minutes and 60minutes. In additional embodiments, this period of time can be greaterthan 120 minutes.

The parameter analyzer 118, the reader 120, or another device incommunication with the parameter analyzer 118 or the reader 120 candetermine the concentration of the infectious agent 102 in the sample104 using measured ORP signals (e.g., measured output voltages) and astandard curve (such as the standard curve 126 described in connectionwith method 100 of FIG. 1). In one example embodiment, a standard curvecan be generated using different concentrations of bacteria (e.g., fromabout 1*10⁴ CFU/mL to about 1*10⁸ CFU/mL) grown at 35° C. in growthmedia. The ORPs of growth media comprising such bacterial concentrationscan be monitored over time for a change in their ORPs using one or moreORP sensors. A threshold voltage can be set (e.g., between about −100 mVand 100 mV) and a standard curve can be generated by plotting thevarious bacterial concentrations against the time it took the monitoredORP of each such bacterial concentration to reach the threshold voltage(also known as the time-to-detection (TTD)). Generation of the standardcurve is discussed in more detail in the following sections.

The reader 120, the parameter analyzer 118, or another device incommunication with either the reader 120 or the parameter analyzer 118can compare the measured or monitored ORP of the diluted sample 112 overtime against the values obtained from the standard curve. The reader120, the parameter analyzer or another device in communication witheither the reader 120 or the parameter analyzer 118 can then determinethe concentration of the infectious agent 102 in the sample 104 underinvestigation by using the time-to-detection and the values obtainedfrom the standard curve. For example, the concentration can becalculated using the time-to-detection and an equation derived from thestandard curve.

In some embodiments, one or more of the aforementioned steps can bestored as machine-executable instructions or logical commands in anon-transitory machine-readable medium (e.g., a memory or storage unit)of the parameter analyzer 118, the reader 120, or another devicecommunicatively or electrically coupled to the parameter analyzer 118 orthe reader 120. Any of the parameter analyzer 118, the reader 120, oranother device coupled to the parameter analyzer 118 or the reader 120can comprise one or more processors or controllers configured to executethe aforementioned instructions or logical commands. In addition, any ofthe devices or systems shown in the example embodiments of FIGS. 2A, 2B,and 2C can be used to perform steps or operations of methods disclosedherein including, but not limited to, methods 100 and 500.

FIG. 3A illustrates bacterial growth curves obtained by monitoring thechange in ORP of growth media comprising different concentrations (e.g.,from about 1*10⁴ CFU/mL to about 1*10⁸ CFU/mL) of a type of bacteria.For example, FIG. 3A illustrates growth curves of differentconcentrations of Pseudomonas aeruginosa (PAe) bacteria grown at 35° C.in Mueller Hinton growth media (MHG). The ORPs of growth media exposedto the various PAe concentrations were monitored using ORP sensors (forexample, any of the sensors 116 of FIGS. 1, 2A, 2B, and 2C). A thresholdvoltage 130 was set at −100 mV and the time it took the monitored ORPsto reach the threshold voltage 130 (i.e., the TTDs 132) were used togenerate the standard curve 126.

FIG. 3B illustrates a standard curve 126 generated using certainexperimental data from the experiments described above. As shown in FIG.3B, a threshold ORP level was set at −100 mV. The various TTDs 132 wereplotted as a function of the logarithm of the known concentration of theinfectious agent 102 present in the various samples. A standard curve126 can then be generated using curve fitting techniques such aslogarithmic regression and least-squares. In other embodiments,polynomial and logarithmic curve fitting techniques can also be used.

As shown in FIG. 3B, a logarithmic standard curve 126 can be generatedusing values obtained from monitoring the ORP of growth media exposed tovarious concentrations of an infectious agent 102. Deriving an equationfor this logarithmic standard curve 126 can then allow us to interpolateunknown concentrations of infectious agents 102 in a sample using onlythe time it took such a solution to reach the ORP threshold voltage 130.

FIG. 4 illustrates bacterial growth curves used in the quantification ofPAe from positive blood cultures. The positive blood cultures wereprepared by adding 10 CFU/mL of PAe to 25 mL of human blood. Theresulting blood comprising PAe was then added to 30 mL of blood culturemedia (e.g., 30 mL of BD BACTEC™ Plus Aerobic Medium). The combinedmixture of human blood containing PAe and blood culture media was thengrown to positivity. Three aliquots of the positive blood culture werethen diluted with growth media to dilution ratios of 1:10, 1:100, and1:1000, respectively. Such diluted samples were then introduced to anORP sensor comprising a redox-active material. FIG. 4 illustrateschanges in the ORP signals of the three diluted samples over time(commonly referred to as bacterial growth curves). As shown in FIG. 4, athreshold voltage of −100 mV was set and the time-to-detection of eachcurve was measured and compared to the PAe standard curve of FIG. 3B.The concentration of the PAe (in CFU/mL) can then be determined usingthe standard curve and by taking into account the amount of dilution.Diluting the positive blood culture with growth media to differentdilution ratios can be helpful in determining the minimum amount ofdilution needed to quantify a certain sample and ensuring that all suchconcentration determinations ultimately align.

FIG. 5 illustrates an embodiment of a method 500 for determining thesusceptibility of one or more infectious agents 102 in a sample 104 toone or more anti-infectives 502. The method 500 can comprise introducingone or more aliquots of the sample 104 into one or more reaction vessels106 in step 5A. The reaction vessels 106 can refer to one or more testtubes, reaction tubes, wells of a high throughput assay plate or wellplate such as a 96-well plate, a 192-well plate, or a 384-well plate,culture plates or dishes, or other suitable containers for housingbiological samples. One or more fluid delivery conduits 108 canintroduce, deliver, or otherwise introduce the aliquots of the sample104 to the one or more reaction vessels 106.

In other embodiments not shown in FIG. 5, a stimulus solution can beadded to the sample 104 before introducing the sample 104 to thereaction vessel 106. The stimulus solution can be a nutrient or growthsolution. In these and other embodiments, the sample 104 can also befiltered before step 5A. This filtering step can involve filtering thesample 104 using an instance of a filter, a microfluidic filter, or acombination thereof to filter out debris, inorganic material, and largercellular components including blood cells or epithelial cells from thesample 104.

The sample 104 can comprise at least one of a biological sample, abodily fluid, a wound swab or sample, a rectal swab or sample, and abacterial culture derived from the biological sample, the bodily fluid,the wound swab or sample, or the rectal swab or sample. The bodily fluidcan comprise urine, blood, serum, plasma, saliva, sputum, semen, breastmilk, joint fluid, spinal fluid, wound material, mucus, fluidaccompanying stool, re-suspended rectal or wound swabs, vaginalsecretions, cerebrospinal fluid, synovial fluid, pleural fluid,peritoneal fluid, pericardial fluid, amniotic fluid, cultures of bodilywhich has been tested positive for bacteria or bacterial growth such asblood culture which has been tested positive for bacteria or bacterialgrowth (i.e., positive blood culture), or a combination thereof.

The infectious agents 102 that can be assayed for anti-infectivesusceptibility using the methods or systems disclosed herein can be anymetabolizing single- or multi-cellular organism including bacteria andfungi. In certain embodiments, the infectious agent 102 can be bacteriaselected from the genera Acinetobacter, Acetobacter, Actinomyces,Aerococcus, Aeromonas, Agrobacterium, Anaplasma, Azorhizobium,Azotobacter, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia,Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Chlamydia,Chlamydophila, Citrobacter, Clostridium, Corynebacterium, Coxiella,Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella,Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella,Lactobacillus, Legionella, Listeria, Methanobacterium, Microbacterium,Micrococcus, Morganella, Moraxella, Mycobacterium, Mycoplasma,Neisseria, Pandoraea, Pasteurella, Peptostreptococcus, Porphyromonas,Prevotella, Proteus, Providencia, Pseudomonas, Ralstonia, Raoultella,Rhizobium, Rickettsia, Rochalimaea, Rothia, Salmonella, Serratia,Shewanella, Shigella, Spirillum, Staphylococcus, Strenotrophomonas,Streptococcus, Streptomyces, Treponema, Vibrio, Wolbachia, Yersinia, ora combination thereof. In other embodiments, the infectious agent 102can be one or more fungi selected from the genera Candida orCryptococcus or mold.

Other specific bacteria that can be assayed for anti-infectivesusceptibility using the methods and systems disclosed herein cancomprise Staphylococcus aureus, Staphylococcus lugdunensis,coagulase-negative Staphylococcus species (including but not limited toStaphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus capitis, not differentiated), Enterococcusfaecalis, Enterococcus faecium (including but not limited toEnterococcus faecium and other Enterococcus spp., not differentiated,excluding Enterococcus faecalis), Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus spp.,(including but not limited to Streptococcus mitis, Streptococcuspyogenes, Streptococcus gallolyticus, Streptococcus agalactiae,Streptococcus pneumoniae, not differentiated), Pseudomonas aeruginosa,Acinetobacter baumannii, Klebsiella spp. (including but not limited toKlebsiella pneumoniae, Klebsiella oxytoca, not differentiated),Escherichia coli, Enterobacter spp. (including but not limited toEnterobacter cloacae, Enterobacter aerogenes, not differentiated),Proteus spp. (including but not limited to Proteus mirabilis, Proteusvulgaris, not differentiated), Citrobacter spp. (including but notlimited to Citrobacter freundii, Citrobacter koseri, notdifferentiated), Serratia marcescens, Candida albicans, and Candidaglabrata.

Other more specific bacteria that can be assayed for anti-infectivesusceptibility can comprise Acinetobacter baumannii, Actinobacillusspp., Actinomycetes, Actinomyces spp. (including but not limited toActinomyces israelii and Actinomyces naeslundii), Aeromonas spp.(including but not limited to Aeromonas hydrophila, Aeromonas veroniibiovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasmaphagocytophilum, Alcaligenes xylosoxidans, Actinobacillusactinomycetemcomitans, Bacillus spp. (including but not limited toBacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillusthuringiensis, and Bacillus stearothermophilus), Bacteroides spp.(including but not limited to Bacteroides fragilis), Bartonella spp.(including but not limited to Bartonella bacilliformis and Bartonellahenselae, Bifidobacterium spp., Bordetella spp. (including but notlimited to Bordetella pertussis, Bordetella parapertussis, andBordetella bronchiseptica), Borrelia spp. (including but not limited toBorrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (includingbut not limited to Brucella abortus, Brucella canis, Brucellamelintensis and Brucella suis), Burkholderia spp. (including but notlimited to Burkholderia pseudomallei and Burkholderia cepacia),Campylobacter spp. (including but not limited to Campylobacter jejuni,Campylobacter coli, Campylobacter lari and Campylobacter fetus),Capnocytophaga spp., Cardiobacterium hominis, Chlamydia trachomatis,Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter spp.Coxiella burnetii, Corynebacterium spp. (including but not limited to,Corynebacterium diphtheriae, Corynebacterium jeikeum andCorynebacterium), Clostridium spp. (including but not limited toClostridium perfringens, Clostridium difficile, Clostridium botulinumand Clostridium tetani), Eikenella corrodens, Enterobacter spp.(including but not limited to Enterobacter aerogenes, Enterobacteragglomerans, Enterobacter cloacae and Escherichia coli, includingopportunistic Escherichia coli, including but not limited toenterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E.coli, enterohemorrhagic E. coli, enteroaggregative E. coli anduropathogenic E. coli) Enterococcus spp. (including but not limited toEnterococcus faecalis and Enterococcus faecium) Ehrlichia spp.(including but not limited to Ehrlichia chafeensia and Ehrlichia canis),Erysipelothrix rhusiopathiae, Eubacterium spp., Francisella tularensis,Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum,Haemophilus spp. (including but not limited to Haemophilus influenzae,Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae,Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacterspp. (including but not limited to Helicobacter pylori, Helicobactercinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella spp.(including but not limited to Klebsiella pneumoniae, Klebsiellagranulomatis and Klebsiella oxytoca), Lactobacillus spp., Listeriamonocytogenes, Leptospira interrogans, Legionella pneumophila,Leptospira interrogans, Peptostreptococcus spp., Moraxella catarrhalis,Morganella spp., Mobiluncus spp., Micrococcus spp., Mycobacterium spp.(including but not limited to Mycobacterium leprae, Mycobacteriumtuberculosis, Mycobacterium intracellulare, Mycobacterium avium,Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm spp.(including but not limited to Mycoplasma pneumoniae, Mycoplasma hominis,and Mycoplasma genitalium), Nocardia spp. (including but not limited toNocardia asteroides, Nocardia cyriacigeorgica and Nocardiabrasiliensis), Neisseria spp. (including but not limited to Neisseriagonorrhoeae and Neisseria meningitidis), Pasteurella multocida,Plesiomonas shigelloides. Prevotella spp., Porphyromonas spp.,Prevotella melaninogenica, Proteus spp. (including but not limited toProteus vulgaris and Proteus mirabilis), Providencia spp. (including butnot limited to Providencia alcalifaciens, Providencia rettgeri andProvidencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes,Rhodococcus equi, Rickettsia spp. (including but not limited toRickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii,Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) andRickettsia typhi), Rhodococcus spp., Serratia marcescens,Stenotrophomonas maltophilia, Salmonella spp. (including but not limitedto Salmonella enterica, Salmonella typhi, Salmonella paratyphi,Salmonella enteritidis, Salmonella cholerasuis and Salmonellatyphimurium), Serratia spp. (including but not limited to Serratiamarcesans and Serratia liquifaciens), Shigella spp. (including but notlimited to Shigella dysenteriae, Shigella flexneri, Shigella boydii andShigella sonnei), Staphylococcus spp. (including but not limited toStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcushemolyticus, Staphylococcus saprophyticus), Streptococcus spp.(including but not limited to Streptococcus pneumoniae (for examplechloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,erythromycin-resistant serotype 14 Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema spp. (includingbut not limited to Treponema carateum, Treponema petenue, Treponemapallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasmaurealyticum, Veillonella sp., Vibrio spp. (including but not limited toVibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrioparahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibriomimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibriodamsela and Vibrio furnisii), Yersinia spp. (including but not limitedto Yersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis) and Xanthomonas maltophilia among others.

Furthermore, other infectious agents 102 that can be assayed foranti-infective susceptibility can comprise fungi or mold including, butnot limited to, Candida spp. (including but not limited to Candidaalbicans, Candida glabrata, Candida tropicalis, Candida parapsilosis,and Candida krusei), Aspergillus spp. (including but not limited toAspergillus fumigatous, Aspergillus flavus, Aspergillus clavatus),Cryptococcous spp. (including but not limited to Cryptococcusneoformans, Cryptococcus gattii, Cryptococcus laurentii, andCryptococcus albidus), Fusarium spp. (including but not limited toFusarium oxysporum, Fusarium solani, Fusarium verticillioides, andFusarium proliferatum), Rhizopus oryzae, Penicillium marneffei,Coccidiodes immitis, and Blastomyces dermatitidis.

The fluid delivery conduits 108 can include tubes, pumps, containers, ormicrofluidic channels for delivering buffers, reagents, fluid samplesincluding the sample 104 or solubilized solutions thereof, othersolutions, or a combination thereof to and between devices, apparatus,or containers in the system. For example, as shown in FIG. 5, the fluiddelivery conduits 108 can refer to parts of a pump such as a syringepump. In other embodiments, the fluid delivery conduits 108 can includeor refer to at least part of a hydraulic pump, a pneumatic pump, aperistaltic pump, a vacuum pump or a positive pressure pump, a manual ormechanical pump, or a combination thereof. In additional embodiments,the fluid delivery conduits 108 can include or refer to at least part ofan injection cartridge, a pipette, a capillary, or a combinationthereof. The fluid delivery conduits 108 can also be part of a vacuumsystem configured to draw fluid to or through channels, tubes, orpassageways under vacuum. Moreover, the fluid delivery conduits 108 caninclude or refer to at least part of a multichannel delivery system orpipette.

The method 500 can comprise diluting the sample 104 comprising the oneor more infectious agents 102 with a dilutive solution 110 to yield adiluted sample 112 in step 5B. In one embodiment, the dilutive solution110 can comprise growth media or a growth inducer. In this and otherembodiments, the dilutive solution 110 can be a solution containingbacto-tryptone, yeast extract, beef extract, cation-adjusted MuellerHinton Broth (CAMHB), Mueller Hinton growth media (MHG), starch, acidhydrolysate of casein, calcium chloride, magnesium chloride, sodiumchloride, blood or lysed blood including lysed horse blood (LHB),CAMHB-LHB, glucose, or a combination thereof. The growth inducer cancomprise a carbon-based inducer, a nitrogen-based inducer, a mineral, atrace element, a biological growth factor, or any combination thereof.For example, the growth inducer can include but is not limited toglucose, ammonia, magnesium, blood, or a combination thereof. In oneexample embodiment, the dilutive solution 110 can comprise Tryptone,yeast extract, sodium chloride, and glucose. The dilutive solution 110can be used to counteract the buffering effects of ions or substancespresent in the sample 104.

In one embodiment, diluting the sample 104 with the dilutive solution110 in step 5B can involve diluting the sample 104 to a dilution ratiobetween about 1:1 to about 1:10. In another embodiment, diluting thesample 104 with the dilutive solution 110 can involve diluting thesample 104 to a dilution ratio between about 1:10 to about 1:100. In yetanother embodiment, diluting the sample 104 with the dilutive solution110 can involve diluting the sample 104 to a dilution ratio betweenabout 1:100 to about 1:1000. In a further embodiment, diluting thesample 104 with the dilutive solution 110 can involve diluting thesample 104 to a dilution ratio between about 1:1000 to about 1:10000.Although FIG. 5 illustrates one reaction vessel 106 or one aliquot ofthe sample 104 being diluted, it is contemplated by this disclosure thatmultiple aliquots of the sample 104 can be diluted to different dilutionratios such that one or more diluted samples 112 can act as internalcontrols.

As will be discussed in the following sections in relation to FIG. 6, inalternative embodiments, the method 500 can comprise diluting the sample104 comprising the infectious agent 102 with deionized water, a salinesolution, or a combination thereof serving as the dilutive solution 110.In these embodiments, the diluted sample(s) 112 can be introduced to oneor more sensors through sample delivery conduits comprising growthmedia/growth inducers and anti-infectives such that the diluted sample112 is mixed with the growth media/growth inducers and anti-infectives.More details concerning these embodiments will be discussed in thefollowing sections.

The method 500 can further comprise separating the diluted sample 112into multiple aliquots such as, for example, a first aliquot and asecond aliquot in step 5C. The method 500 can also comprise introducingand mixing an anti-infective 502 at a first concentration into the firstaliquot of the diluted sample 112. The mixture comprising the firstaliquot and the anti-infective 502 at the first concentration can bereferred to as a test solution 506. The second aliquot of the dilutedsample 112 without the anti-infective 502 can be used as a controlsolution 504. Although FIG. 5 illustrates only one test solution 506comprising the first aliquot and the anti-infective 502, it iscontemplated by this disclosure and should be understood by one ofordinary skill in the art that the method 500 and systems disclosedherein can assay multiple test solutions and some such test solutionscan comprise a different anti-infective 502, the same anti-infective 502at a different concentration, or a different anti-infective 502 at adifferent concentration. For example, the anti-infective 502 can bediluted to two different concentrations before being introduced to twodifferent reaction vessels 106 containing aliquots of the diluted sample112.

The anti-infective 502 used in the systems and methods disclosed hereincan comprise a bacteriostatic anti-infective, a bactericidalanti-infective, an anti-fungal anti-infective, or a combination thereof.

In certain embodiments, the bacteriostatic anti-infective can compriseβ-lactams (including but not limited to penicillins such as ampicillin,amoxicillin, flucloxacillin, penicillin, amoxicillin/clavulanate, andticarcillin/clavulanate and monobactams such as aztreonam), β-lactam andβ-lactam inhibitor combinations (including but not limited topiperacillin-tazobactam and ampicillin-sulbactam), Aminoglycosides(including but not limited to amikacin, gentamicin, kanamycin, neomycin,netilmicin, paromomycin, streptomycin, spectinomycin, and tobramycin),Ansamycins (including but not limited to rifaximin), Carbapenems(including but not limited to ertapenem, doripenem, imipenem, andmeropenem), Cephalosporins (including but not limited to ceftaroline,cefepime, ceftazidime, ceftriaxone, cefadroxil, cefalotin, cefazolin,cephalexin, cefaclor, cefprozil, fecluroxime, cefixime, cefdinir,cefditoren, cefotaxime, cefpodoxime, ceftibuten, and ceftobiprole),Chloramphenicols, Glycopeptides (including but not limited tovancomycin, teicoplanin, telavancin, dalbavancin, and oritavancin),Folate Synthesis Inhibitors (including but not limited totrimethoprim-sulfamethoxazole), Fluoroquinolones (including but notlimited to ciprofloxacin), Lincosamides (including but not limited toclindamycin, lincomycin, azithromycin, clarithromycin, dirithromycin,roxithromycin, telithromycin, and spiramycin), Lincosamines,Lipopeptides, Macrolides (including but not limited to erythromycin),Monobactams, Nitrofurans (including but not limited to furazolidone andnitrofurantoin), Oxazolidinones (including but not limited to linezolid,posizolid, radezolid, and torezolid), Quinolones (including but notlimited to enoxacin, gatifloxacin, gemifloxacin, levofloxacin,lomefloxacin, moxifloxacin, naldixic acid, norfloxacin, trovafloxacin,grepafloxacin, sparfloxacin, and temafloxacin), Rifampins,Streptogramins, Sulfonamides (including but not limited to mafenide,sulfacetamide, sulfadiazine, sulfadimethoxine, sulfamethizole,sulfamethoxazole, sulfasalazine, and sulfisoxazole), Tetracyclines(including but not limited to oxycycline, minocycline, demeclocycline,doxycycline, oxytetracycline, and tetracycline), polypeptides (includingbut not limited to bacitracin, polymyxin B, colistin, and cycliclipopeptides such as daptomycin), phages, or a combination or derivativethereof.

In other embodiments, the anti-infective 502 can comprise clofazimine,ethambutol, isoniazid, rifampicin, arsphenamine, chloramphenicol,fosfomycin, metronidazole, tigecycline, trimethoprim, or a combinationor derivative thereof.

In certain embodiments, the anti-fungal can comprise Amphotericin B,Anidulafungin, Caspofungin, Fluconazole, Flucytosine, Itraconazole,Ketoconazole, Micafungin, Posaconazole, Ravuconazole, Voriconazole, or acombination or derivative thereof.

The method 500 can also comprise incubating the first aliquot and thesecond aliquot at an elevated temperature for a period of time in step5E. The first aliquot and the second aliquot can be incubated in theirrespective reaction vessels 106 or transferred to different reactionvessels 106 or containers. For example, the first aliquot and the secondaliquot can be heated to a temperature of between about 30° C. and about40° C. (e.g., 35° C.±2° C.) and allowed to incubate for an incubationperiod 114. The incubation period 114 can range from 15 minutes to overone hour. In other embodiments, the incubation period 114 can be lessthan 15 minutes or up to 48 hours.

The incubation period 114 can be adjusted based on the type ofinfectious agent 102 suspected in the sample 104, such as the type ofbacteria or fungus. The incubation period 114 can also be adjusted basedon the type of anti-infective 502, the mechanism of action of theanti-infective 502, the amount of the sample 104, or a combinationthereof. The incubation period 114 can be start-delayed or apre-incubation time period can be added before the start of theincubation period 114. The start-delay or the pre-incubation time periodcan be added for slower acting drugs or anti-infectives 502 (e.g.,β-lactams). In some embodiments, the start-delay or the pre-incubationtime period can be between 10 minutes and 2 hours. In other embodiments,the start-delay or the pre-incubation time period can be as long asneeded for the drug or anti-infective 502 to take effect. During thestart-delay or pre-incubation time period, readings or measurements fromthe sensor(s) would not be used or would not be included as part of anygrowth curves generated (ORP signals monitored). The start-delay or thepre-incubation time period is particularly useful for instances wherehigher inoculums or a higher concentration of infectious agents 102 ispresent in the sample 104 or aliquots and where the signal is generatedrelatively fast in comparison to the mode of action of the drug oranti-infective 502.

The method 500 can further comprise introducing the test solution 506 toa first sensor 508 or exposing the first sensor 508 to the test solution506 such that the test solution 506 is in fluid communication with aredox-active material of the first sensor 508 in step 5F(i). The method500 can also comprise introducing the control solution 504 to a secondsensor 510 or exposing the second sensor 510 to the control solution 504such that the control solution 504 is in fluid communication with theredox-active material of the second sensor 510 in step 5F(ii).

In certain embodiments, the first sensor 508 and the second sensor 510can be oxidation reduction potential (ORP) sensors configured to respondto a change in a solution characteristic (e.g., the ORP) of a measuredsolution. In the example embodiment shown in FIG. 5, exposing the firstsensor 508 and the second sensor 510 to the test solution 506 and thecontrol solution 504, respectively, can involve directly immersing atleast part of a handheld or probe instance of the first sensor 508 andthe second sensor 510 into the test solution 506 and the controlsolution 504, respectively. In this embodiment, the handheld or probeinstance of the first sensor 508 or the second sensor 510 can be ahandheld OPR sensor coupled to a standalone parameter analyzer 118 suchas a voltmeter or multimeter. In alternative example embodiments alsoshown in FIG. 5, introducing the test solution 506 and the controlsolution 504 to the first sensor 508 and the second sensor 510,respectively, can involve injecting, delivering, or otherwiseintroducing the test solution 506 to a well or container comprising thefirst sensor 508 and introducing the control solution 504 to anotherwell or container comprising the second sensor 510. In theseembodiments, the first sensor 508 and the second sensor 510 can befabricated on one substrate 202 or different substrates 202.

The substrate 202 can be comprised of a polymeric material, a metal, aceramic, a semiconductor layer, an oxide layer, an insulator, or acombination thereof. The substrate 202 can be part of a test strip,cartridge, chip or lab-on-a-chip, microfluidic device, multi-wellcontainer, or a combination thereof. In some embodiments, the one ormore parameter analyzers 118 can also be fabricated or located on thesubstrate 202. In other embodiments, the one or more parameter analyzers118 can be standalone devices such as a voltmeter or a multimeterelectrically coupled to the sensors.

As will be discussed in more detail in the following sections, each ofthe first sensor 508 and the second sensor 510 can comprise an activeelectrode and a reference electrode. In addition, the redox-activematerial 908 can comprise a gold layer, a platinum layer, a metal oxidelayer, a carbon layer, or a combination thereof.

The method 500 can further comprise monitoring the ORP of the testsolution 506 over a period of time using one or more parameter analyzers118 coupled to the first sensor 508 in step 5F(i). The method 500 canalso comprise monitoring the ORP of the control solution 504 over asimilar period of time using one or more parameter analyzers 118 coupledto the second sensor 510 in step 5F(ii). In one or more embodiments, theORPs of the test solution 506 and the control solution 504 can bemonitored in the absence of any added or exogenous reporter moleculespresent in the test solution 506 or the control solution 504.

The test solution 506 and the control solution 504 can each have asolution characteristic. The solution characteristic of the testsolution 506 and the solution characteristic of the control solution 504can change as the amount of electro-active redox species changes due tothe energy use, oxygen uptake or release, growth, or lack thereof of theinfectious agents 102 in the test solution 506 and the control solution504. For example, the amount of electro-active redox species in the testsolution 506 can change as a result of increasing or diminishingcellular activity undertaken by the infectious agents 102 in the testsolution 506. Also, for example, the amount of electro-active redoxspecies in the control solution 504 can change as a result of cellularactivity undertaken by the infectious agents 102 in the control solution504. As a more specific example, the amount of electron donors fromTable 1 (e.g., the amount of energy carriers such as nicotinamideadenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH₂)) inthe test solution 506 or the control solution 504 can change due to thegrowth or lack thereof of the infectious agents 102 in the test solution506 or the control solution 504. Also, as another more specific example,the amount of oxygen depleted in the test solution 506 or the controlsolution 504 due to aerobic respiration can change due to the growth orlack thereof of the infectious agents 102 in the test solution 506 orthe control solution 504.

The method 500 can further comprise comparing the ORP of the testsolution 506 with the ORP of the control solution 504 to determine thesusceptibility of the infectious agent 102 to the anti-infective 502 instep 5G. In some embodiments, comparing the ORP of the test solution 506with the ORP of the control solution 504 can be done using one or moreparameter analyzers 118 coupled to the first sensor 508, the secondsensor 510, or a combination thereof. In other embodiments, comparingthe ORP of the test solution 506 with the ORP of the control solution504 can be done using another device electrically or communicativelycoupled to the parameter analyzer 118 such as the reader 120. In yetadditional embodiments, comparing the ORP of the test solution 506 withthe ORP of the control solution 504 can be done using a combination ofone or more parameter analyzers 118 and the reader 120.

In certain embodiments, the reader 120 can be a mobile device, ahandheld device, a tablet device, or a computing device such as a laptopor desktop computer having a display 122. For example, the display 122can be a mobile device display, a handheld device display, a tabletdisplay, or a laptop or desktop monitor. In some embodiments, theparameter analyzer 118 can also comprise a display or can wirelesslycommunicate a signal or readout to a device having a display.

The parameter analyzer 118, the reader 120, or a combination thereof canmonitor and compare the ORP of the test solution 506 with the ORP of thecontrol solution 504 over a period of time. The period of time can bereferred to as a detection window 512. The parameter analyzer 118, thereader 120, or a combination thereof can assess the susceptibility ofthe infectious agent 102 to the anti-infective 502 within this detectionwindow 512. In one embodiment, the detection window 512 can be between60 minutes and 120 minutes. In other embodiments, the detection window512 can be between 5 minutes and 60 minutes. In additional embodiments,the detection window 512 can be greater than 120 minutes.

In one embodiment, the parameter analyzer 118, the reader 120, or acombination thereof can comprise one or more controllers or processorsto execute logical commands concerning the comparison of the ORP of thetest solution 506 with the ORP of the control solution 504. In this andother embodiments, the parameter analyzer 118, the reader 120, or acombination thereof can generate or instruct another device to generatea read-out, graph, or signal concerning a result of the comparison on adisplay such as the display 122.

For example, the parameter analyzer 118, the reader 120, or acombination thereof can determine or assess the susceptibility of theinfectious agent 102 in the sample 104 as resistant to an anti-infective502 when the parameter analyzer 118, the reader 120, or a combinationthereof fails to detect a statistically significant difference betweenthe ORP of the test solution 506 and the ORP of the control solution504. This statistically significant difference can be a differenceexceeding a threshold value or range. Conversely, the parameter analyzer118, the reader 120, or a combination thereof can determine or assessthe susceptibility of the infectious agent 102 as susceptible to ananti-infective 502 when the parameter analyzer 118, the reader 120, or acombination thereof detects certain statistically significantdifferences between the ORP of the test solution 506 and the ORP of thecontrol solution 504 within the detection window 512.

Although not shown in FIG. 5, the method 500 can also compriseseparating the diluted sample 112 into a third aliquot and introducingthe anti-infective 502 at a second concentration into the third aliquotto form another test solution. In some embodiments, the secondconcentration of the anti-infective 502 can be less than the firstconcentration of the anti-infective 502 added to the first aliquot. Inthese embodiments, the second concentration of the anti-infective 502can be obtained by diluting the first concentration of theanti-infective 502. In other embodiments, the second concentration canbe greater than the first concentration.

The method 500 can further comprise introducing the other test solutionto a third sensor such that the other test solution is in fluidcommunication with the redox-active material of the third sensor. TheORP of the other test solution 506 can be monitored over a period oftime using one or more parameter analyzers 118 coupled to the thirdsensor. The ORP can be monitored in the absence of any added reportermolecules in the other test solution. The method 500 can also comprisecomparing the ORP of the other test solution with the ORPs of the testsolution 506 formed from the first aliquot and the control solution 504.The ORPs can be compared to determine a degree of susceptibility of theinfectious agent 102 to the anti-infective 502. For example, theparameter analyzer 118, the reader 120, or a combination thereof canassess the degree or level of susceptibility of the infectious agent 102in the sample 104 on a tiered scale. As a more specific example, theparameter analyzer 118, the reader 120, or a combination thereof canassess the susceptibility of the infectious agent 102 in the sample 104as being resistant, of intermediate susceptibility, or susceptible tothe anti-infective 502 based on a comparison of the ORPs of the two testsolutions with each other and comparisons of the ORPs of the two testsolutions with the control solution 504.

In some embodiments, one or more of the aforementioned steps of themethod 500 can be stored as machine-executable instructions or logicalcommands in a non-transitory machine-readable medium (e.g., a memory orstorage unit) of the parameter analyzer 118, the reader 120, or anotherdevice communicatively or electrically coupled to the parameter analyzer118 or the reader 120. Any of the parameter analyzer 118, the reader120, or another device coupled to the parameter analyzer 118 or thereader 120 can comprise one or more processors or controllers configuredto execute the aforementioned instructions or logical commands.

The steps depicted in FIG. 5 do not require the particular order shownto achieve the desired result. Moreover, certain steps or processes maybe omitted or occur in parallel in order to achieve the desired result.In addition, any of the systems or devices disclosed herein can be usedin lieu of devices or systems shown in the steps of FIG. 5.

FIG. 6 illustrates an embodiment of a multiplex system 600 fordetermining a susceptibility an infectious agent 102 as well as a levelof susceptibility of an infectious agent 102 to one or moreanti-infectives 502. In some embodiments, the multiplex system 600 canbe part of a cartridge, a test strip, an integrated circuit, amicro-electro-mechanical system (MEMS) device, a microfluidic system orchip, or a combination thereof.

The system 600 can be another embodiment of the system 200 illustratedin FIG. 2C with many of the same components as the system 200. Thesystem 600 can comprise the same sample delivery surface 204 defined onthe same substrate 202. In one or more embodiments, the sample receivingsurface 204 can be a flat surface for receiving the sample 104. In otherembodiments, the sample receiving surface 204 can be a concave ortapered surface of a well, divot, dish, or container. For example, thesample 104 can be injected, pipetted, pumped, spotted, or otherwiseintroduced to the sample receiving surface 204 for analysis.

The system 600 can also comprise the one or more metering conduits 206in fluid communication with the sample receiving surface 204. In someembodiments, the one or more metering conduits 206 can be channels,passageways, capillaries, tubes, parts therein, or combinations thereoffor delivering the dilutive solution 110 to the sample 104 on the samplereceiving surface 204. For example, the one or more metering conduits206 can refer to channels, passageways, capillaries, or tubes defined onthe substrate 202. Also, for example, the one or more metering conduits206 can refer to channels, passageways, capillaries, or tubes serving aspart of hydraulic pump, a pneumatic pump, peristaltic pump, a vacuum orpositive pressure pump, a manual or mechanical pump, a syringe pump, ora combination thereof. For example, the one or more metering conduits206 can be microfluidic channels or tubes or channels serving as part ofa vacuum system.

In some embodiments, the one or more metering conduits 206 can beconfigured to dilute the sample 104 with the dilutive solution 110 to adilution ratio between about 1:1 to about 1:10. In other embodiments,the one or more metering conduits 206 can be configured to dilute thesample 104 with the dilutive solution 110 to a dilution ratio betweenabout 1:10 to about 1:100. In additional embodiments, the one or moremetering conduits 206 can be configured to dilute the sample 104 withthe dilutive solution 110 to a dilution ratio between about 1:100 toabout 1:1000. In yet additional embodiments, the one or more meteringconduits 206 can be configured to dilute the sample 104 with thedilutive solution 110 to a dilution ratio between about 1:1000 to about1:10000.

The system 600 can also comprise a plurality of sensors and a pluralityof sample delivery conduits connecting and extending in between each ofthe sensors and the sample receiving surface 204, the one or moremetering conduits 206, or a combination thereof. In certain embodiments,the one or more metering conduits 206 can also separate the dilutedsample into multiple aliquots including at least a first aliquot, asecond aliquot, a third aliquot, a fourth aliquot, and a fifth aliquot.In these embodiments, aliquots of the diluted sample can automaticallyflow from the one or more metering conduits 206 into the sample deliveryconduits leading to the sensors.

In other embodiments, the sample 104 can be diluted by a user ortechnician in a separate reaction vessel, test tube, or container. Inthese embodiments, the user can separate the diluted sample intomultiple aliquots and introduce each of the aliquots to either thesample delivery conduits or the sensors directly.

In the example embodiment shown in FIG. 6, the plurality of sensors cancomprise at least the first sensor 508, the second sensor 510, a thirdsensor 602, a fourth sensor 604, and a fifth sensor 606. Although fivesensors are described herein it should be understood by one of ordinaryskill in the art that the system 600 can comprise more than fivesensors.

In some embodiments, the sensors (including any of the first sensor 508,the second sensor 510, the third sensor 602, the fourth sensor 604, andthe fifth sensor 606) can be the sensors 900 described in connectionwith FIGS. 9A and 9B. For example, the sensors can be micro- ornano-scale ORP sensors. The sensors can be fabricated or located on asurface of the substrate 202. For example, the substrate 202 can be partof a circuit, chip, or device and the sensors can comprise components ofsuch circuits, chips, or devices including, but not limited to, one ormore transistors, gates, or other electrical components. In someembodiments, the sensors can be positioned within a well, divot,cut-out, or groove defined along the substrate 202. In these and otherembodiments, the diluted samples can be injected, directed, or otherwiseintroduced into each of the wells, divots, cut-outs, or grooves.

Each of the sensors can comprise an active electrode and a referenceelectrode. Each of the sensors can also comprise a redox-active material908 (see FIGS. 9A and 9B) or layer such as a gold layer, a platinumlayer, a metal oxide layer, carbon layer, or a combination thereof. Thesensors will be discussed in more detail in the following sections.

The sample delivery conduits (e.g., the first sample delivery conduit608, the second sample delivery conduit 610, the third sample deliveryconduit 612, the fourth sample delivery conduit 614, and the fifthsample delivery conduit 616) can extend in between the sample receivingsurface 204 and the plurality of sensors or extend in between the one ormore metering conduits 206 and the plurality of sensors. The sampledelivery conduits can be channels, passageways, capillaries, tubes,microfluidic channels, parts therein, or combinations thereof fordelivering the diluted sample to the sensors. The sample deliveryconduits can allow aliquots of the diluted sample to be in fluidcommunication the sensors. For example, each of the sample deliveryconduits can allow an aliquot of the diluted ample to be in fluidcommunication with a redox-active material or layer of a sensor.

In the example embodiment shown in FIG. 6, each of the sample deliveryconduits can be covered or coated by a lyophilized or dried form of ananti-infective. The anti-infective can be any of the anti-infectives 502discussed in connection with FIG. 5. The sample delivery conduits can beconfigured such that aliquots of the diluted sample flow through thesample delivery conduits and mix with the lyophilized or dried forms ofthe anti-infective en route to the sensors. In this and other exampleembodiments, the dilutive solution 110 used to dilute the sample 104 cancomprise growth media such as Mueller Hinton growth media (MHG), agrowth inducer, or a combination thereof.

In other embodiments, the dilutive solution 110 used to dilute thesample 104 can be deionized water or saline solution and the sampledelivery conduits 208 can be covered or coated by both a lyophilized ordried form of the anti-infective and a lyophilized or dried form of thegrowth media. In these embodiments, aliquots of the diluted sampleflowing through the sample delivery conduits can mix with thelyophilized or dried forms of the anti-infective and the growth media enroute to the sensors.

In additional embodiments not shown in FIG. 6, the sample deliveryconduits 208 can contain anti-infectives, growth media, or a combinationthereof in aqueous form. In these embodiments, aliquots of the dilutedsample can mix with the aqueous forms of the anti-infective, the growthmedia, or a combination thereof en route to the sensors.

In yet additional embodiments, some of the sensors themselves (e.g., oneor more layers of the sensor) can be covered or coated by lyophilized ordried forms of the anti-infective, the growth media, or a combinationthereof. In these embodiments, aliquots of the diluted sample can mixwith the anti-infective, the growth media, or a combination thereof whenthe aliquots reach or are in fluid communication with the sensors.Moreover, in other embodiments not shown in the figures, a surface inthe vicinity of the sensors can be covered or coated by lyophilized ordried forms of the anti-infective, the growth media, or a combinationthereof. In these embodiments, aliquots of the diluted sample can mixwith the lyophilized or dried forms of the anti-infective, the growthmedia, or a combination thereof when the diluted sample is in fluidcommunication with the surface covered or coated by the lyophilizedanti-infective or growth media.

In all such embodiments, at least one of the sample delivery conduitsleading up to at least one of the sensors can be free or devoid ofanti-infectives. In these embodiments, the diluted sample flowingthrough this sample delivery conduit can act as a control solution.Also, in these embodiments, each of the aliquots of the diluted samplemixed with the anti-infective can be referred to as a test solution.

The system 600 shown in FIG. 6 can be used to determine thesusceptibility of a sample 104 comprising the infectious agent 102 tomultiple anti-infectives (as well as multiple concentrations of one ormore anti-infectives) concurrently. As such, one benefit of themultiplex system 600 of FIG. 6 is the ability to perform high-throughputantibiotic susceptibility testing.

In further alternative embodiments not shown in the figures, a user candilute the sample 104 with growth media and mix one or moreanti-infectives into aliquots of the diluted sample prior to introducingthe mixture to the system 600. In these embodiments, the user canintroduce the mixture comprising the diluted sample and theanti-infectives to the sample receiving surface 204 or the sensorsdirectly.

In some embodiments, the system 600 can further comprise an incubatingcomponent configured to incubate the diluted sample mixed with theanti-infective, the growth media, or a combination thereof by heatingthe mixture to a temperature of between about 30° C. and about 40° C.(e.g., 35° C.±2° C.) for a period of time (e.g., the incubation period114).

The system 600 can also comprise one or more parameter analyzers 118electrically or communicatively coupled to the sensors and a reader 120electrically or communicatively coupled to the one or more parameteranalyzers 118. In some embodiments, the one or more parameter analyzers118 can be fabricated or located on the substrate 202. In otherembodiments, the one or more parameter analyzers 118 can be standalonedevices such as a voltmeter or a multimeter electrically coupled to thesensor. In some embodiments, the reader 120 and the parameteranalyzer(s) 118 can be integrated into one device. The parameteranalyzer 118 and the reader 120 depicted in FIG. 6 can be the sameparameter analyzers 118 and reader 120 depicted in FIG. 5.

The parameter analyzer 118, the reader 120, or a combination thereof canmonitor and compare the ORP of the test solution with the ORP of one ormore control solutions over a period of time. This period of time can bebetween 60 minutes and 120 minutes. In other embodiments, this period oftime can be between 5 minutes and 60 minutes. In additional embodiments,this period of time can be greater than 120 minutes.

In some embodiments, the parameter analyzer 118, the reader 120, or acombination thereof can comprise one or more controllers or processorsto execute logical commands concerning the comparison of the ORPs of thetest solutions with the ORP of the control solution. In this and otherembodiments, the parameter analyzer 118, the reader 120, or acombination thereof can generate or instruct another device to generatea read-out, graph, or signal concerning a result of the comparison on adisplay such as the display 122.

For example, the parameter analyzer 118, the reader 120, or acombination thereof can determine or assess the susceptibility of theinfectious agent 102 in the sample 104 as resistant to an anti-infectivewhen the parameter analyzer 118, the reader 120, or a combinationthereof fails to detect a statistically significant difference betweenthe ORP of one of the test solutions and the ORP of the controlsolution. This statistically significant difference can be a differenceexceeding a threshold value or range. Conversely, the parameter analyzer118, the reader 120, or a combination thereof can determine or assessthe susceptibility of the infectious agent 102 as susceptible to ananti-infective when the parameter analyzer 118, the reader 120, or acombination thereof detects certain statistically significantdifferences between the ORP of one of the test solutions and the ORP ofthe control solution.

As will be discussed in the following sections, the system 600 can alsoassess the degree or level of susceptibility of the infectious agent 102in the sample 104 on a tiered scale. As a more specific example, theparameter analyzer 118, the reader 120, or a combination thereof canassess the susceptibility of the infectious agent 102 in the sample 104as being resistant, of intermediate susceptibility, or susceptible tothe anti-infective 502 based on a comparison of the ORPs of two testsolutions with each other and comparisons of the ORPs of the two testsolutions with the control solution 504.

For example, as shown in FIG. 6, the system 600 can comprise at least afirst sample delivery conduit 608, a second sample delivery conduit 610,a third sample delivery conduit 612, a fourth sample delivery conduit614, and a fifth sample delivery conduit 616. The metering conduit 206can also separate the diluted sample into a first aliquot, a secondaliquot, a third aliquot, a fourth aliquot, and a fifth aliquot. Thesystem 600 can direct the first aliquot to the first sample deliveryconduit 608, the second aliquot to the second sample delivery conduit610, the third aliquot to the third sample delivery conduit 612, thefourth aliquot to the fourth sample delivery conduit 614, and the fifthaliquot to the fifth sample delivery conduit 616.

The first sample delivery conduit 608 can comprise a firstanti-infective at a first concentration and the third sample deliveryconduit 612 can comprise the first anti-infective at a secondconcentration. In some embodiments, the second concentration can be lessthan the first concentration and can be obtained by diluting a solutioncomprising the first anti-infective at the first concentration.

The fourth sample delivery conduit 614 can comprise a secondanti-infective at a first concentration and the fifth sample deliveryconduit 616 can comprise the second anti-infective at a secondconcentration. The second anti-infective can be a differentanti-infective than the first anti-infective.

The second sample delivery conduit 610 can be free or devoid of anyanti-infective such that the second aliquot of the diluted sampleintroduced through the second sample delivery conduit 610 can beconsidered a control solution. The first sample delivery conduit 608 canbe configured to introduce the first aliquot of the diluted sample tothe first sensor 508. The first aliquot can mix with the lyophilized ordried first anti-infective at the first concentration to form a firsttest solution. The third sample delivery conduit 612 can be configuredto introduce the third aliquot of the diluted sample to the third sensor602. The third aliquot can mix with the lyophilized or dried firstanti-infective at the second concentration to form a second testsolution. The fourth sample delivery conduit 614 can be configured tointroduce the fourth aliquot of the diluted sample to the fourth sensor604. The fourth aliquot can mix with the lyophilized or dried secondanti-infective at the first concentration to form a third test solution.The fifth sample delivery conduit 616 can be configured to introduce thefifth aliquot of the diluted sample to the fifth sensor 606. The fifthaliquot can mix with the lyophilized or dried second anti-infective atthe second concentration to form a fourth test solution.

The parameter analyzer 118, the reader 120, or a combination thereof canmonitor the ORP of the first test solution when the first test solutionis in fluid communication with the redox-active material of the firstsensor 508. The parameter analyzer 118, the reader 120, or a combinationthereof can monitor the ORP of the control solution when the controlsolution is in fluid communication with the redox-active material of thesecond sensor 510. The parameter analyzer 118, the reader 120, or acombination thereof can monitor the ORP of the second test solution whenthe second test solution is in fluid communication with the redox-activematerial of the third sensor 602. The parameter analyzer 118, the reader120, or a combination thereof can monitor the ORP of the third testsolution when the third test solution is in fluid communication with theredox-active material of the fourth sensor 604. The parameter analyzer118, the reader 120, or a combination thereof can monitor the ORP of thefourth test solution when the fourth test solution is in fluidcommunication with the redox-active material of the fifth sensor 606.The ORPs of the first test solution, the second test solution, the thirdtest solution, the fourth test solution, and the control solution can bemonitored in the absence of any added reporter or exogenous reportermolecules in the first test solution, the second test solution, thethird test solution, the fourth test solution, and the control solution,respectively.

The parameter analyzer 118, the reader 120, or a combination thereof cancompare the ORP of the second test solution with the ORPs of the firsttest solution and the control solution to determine a degree ofsusceptibility of the infectious agent 102 to the first anti-infective.For example, the parameter analyzer 118, the reader 120, or acombination thereof can determine the infectious agent 102 assusceptible to the first anti-infective when the parameter analyzer 118,the reader 120, or a combination thereof detects both a statisticallysignificant difference between the ORP of the first test solution andthe ORP of the control solution (i.e., the infectious agents 102 aredead or dying in the first test solution) and a statisticallysignificant difference between the ORP of the second test solution andthe ORP of the control solution (i.e., the infectious agents 102 aredead or dying in the second test solution). Alternatively, the parameteranalyzer 118, the reader 120, or a combination thereof can determine theinfectious agent 102 as resistant to the first anti-infective when theparameter analyzer 118, the reader 120, or a combination thereof failsto detect a statistically significant difference between the ORP of thefirst test solution and the ORP of the control solution (i.e., theinfectious agents 102 are alive and growing in the first test solution)and fails to detect a statistically significant difference between theORP of the second test solution and the ORP of the control solution(i.e., the infectious agents 102 are alive and growing in the secondtest solution). As a further alternative example, the parameter analyzer118, the reader 120, or a combination thereof can determine theinfectious agent 102 as of intermediate susceptibility to the firstanti-infective when the parameter analyzer 118, the reader 120, or acombination thereof detects a statistically significant differencebetween the ORP of the first test solution and the ORP of the controlsolution (i.e., the infectious agents 102 are dead or dying in the firsttest solution or the first anti-infective at a higher concentration) butfails to detect a statistically significant difference between the ORPof the second test solution and the ORP of the control solution (i.e.,the infectious agents 102 are alive and growing in the second testsolution or the first anti-infective at the lower concentration).

The parameter analyzer 118, the reader 120, or a combination thereof canalso compare the ORP of the fourth test solution with the ORPs of thethird test solution and the control solution to determine a degree ofsusceptibility of the infectious agent 102 to the second anti-infective.For example, the parameter analyzer 118, the reader 120, or acombination thereof can determine the infectious agent 102 assusceptible to the second anti-infective when the parameter analyzer118, the reader 120, or a combination thereof detects both astatistically significant difference between the ORP of the third testsolution and the ORP of the control solution (i.e., the infectiousagents 102 are dead or dying in the third test solution (which is thesecond anti-infective at the higher concentration)) and a statisticallysignificant difference between the ORP of the fourth test solution andthe ORP of the control solution (i.e., the infectious agents 102 aredead or dying in the fourth test solution (which is the secondanti-infective at the lower concentration)). Alternatively, theparameter analyzer 118, the reader 120, or a combination thereof candetermine the infectious agent 102 as resistant to the secondanti-infective when the parameter analyzer 118, the reader 120, or acombination thereof fails to detect a statistically significantdifference between the ORP of the third test solution and the ORP of thecontrol solution (i.e., the infectious agents 102 are alive and growingin the third test solution (which is the second anti-infective at thehigher concentration)) and fails to detect a statistically significantdifference between the ORP of the fourth test solution and the ORP ofthe control solution (i.e., the infectious agents 102 are alive andgrowing in the fourth test solution (which is the second anti-infectiveat the lower concentration)). Furthermore, the parameter analyzer 118,the reader 120, or a combination thereof can determine the infectiousagent 102 as of intermediate susceptibility to the second anti-infectivewhen the parameter analyzer 118, the reader 120, or a combinationthereof detects a statistically significant difference between the ORPof the third test solution and the ORP of the control solution (i.e.,the infectious agents 102 are dead or dying in the third test solution(which is the second anti-infective at the higher concentration)) butfails to detect a statistically significant difference between the ORPof the fourth test solution and the ORP of the control solution (i.e.,the infectious agents 102 are alive and growing in the fourth testsolution (which is the second anti-infective at the lowerconcentration)).

FIG. 7A illustrates an example growth curve 700 of an infectious agent102 not susceptible or resistant to an anti-infective (such asanti-infective 502) in solution. The growth curve 700 can be recorded bymonitoring the sensor output of an ORP sensor (including, but notlimited to, the first sensor 508 or the second sensor 510) in fluidcommunication with the sampled solution. In one embodiment, the sensoroutput can be a potential difference between an active electrode and areference electrode (see FIGS. 9A and 9B). The sensor output of the ORPsensor can change as the ORP of the sampled solution (e.g., any of thetest solutions or the control solution 504) changes.

The voltage output of the ORP sensor can change over time. For example,as shown in FIG. 7A, the voltage output of the sensor can decrease overtime as the solution characteristic of the sampled solution changes dueto the energy use, oxygen uptake or release, growth, or metabolism ofthe infectious agents 102 in solution. In some embodiments, the change(e.g., decrease) in the voltage output of the sensor can follow asigmoidal pattern or shape, a step function or shape, or other patternsor shapes. Over longer time scales, the sensor output or voltage canbegin to increase or become more positive.

For example, the voltage output of the sensor can decrease over time asthe solution characteristic of the sampled solution changes as a resultof cellular activity undertaken by the infectious agents 102 insolution. As a more specific example, the solution characteristic of thesampled solution can change as the amount of energy carriers (such asnicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide(FADH₂)) in the sampled solution changes due to the growth ofanti-infective resistant infectious agents 102. Also, as another morespecific example, the amount of oxygen depleted in the sampled solutioncan change due to the growth or lack thereof of the infectious agents102 in solution.

FIG. 7B illustrates an example growth curve 702 of an infectious agent102 susceptible to or not resistant to an anti-infective (such asanti-infective 502) in solution. The growth curve 702 can be recorded bymonitoring the sensor output of an ORP sensor in fluid communicationwith the sampled solution. As shown in FIG. 7B, the growth curve 702 canbe relatively constant (e.g., a substantially flat line) or change verylittle. In other embodiments not shown in FIG. 7B, the growth curve 702can exhibit changes within a predetermined threshold range. The sensoroutput of the ORP sensor can stay relatively constant as the ORP of thesampled solution (e.g., any of the test solutions or the controlsolution 504) stays relatively constant.

In one embodiment, the voltage output of the ORP sensor can be apotential difference between an active electrode and a referenceelectrode such as the external reference electrode, the on-chipreference electrode, or another reference electrode.

The voltage output of the ORP sensor can stay relatively constant as thesolution characteristic of the sampled solution stays relativelyconstant due to the inhibitive effects of the anti-infective 502 on theinfectious agents 102 in solution.

FIG. 8 illustrates example growth curves of Pseudomonas aeruginosa (PAe)from positive blood culture in the presence of various anti-infectives502. Blood culture positive for PAe was diluted into Mueller Hintongrowth media (MHG) to a concentration of 5*10⁵ CFU/mL and probed withdifferent antibiotics at their susceptibility breakpoints. As shown inFIG. 8, the antibiotics include (1) imipenem (IMI), (2) ceftazidime(CAZ), (3) doripenem (DOR), (4) cefepime (CPM), (5) levofloxacin (LVX),(6) ciprofloxacin (CIP), (7) norfloxacin (NOR), and (8) gentamicin(GEN). PAe and antibiotic mixtures were exposed to ORP sensors (forexample, any of the sensors discussed in connection with FIGS. 5 and 6)and changes in the ORP of the mixture were assessed over time andcompared to the bacterial sample without antibiotic (curve labeled MHGin FIG. 8). A flat or substantially flat line over the entire detectionperiod can indicate elimination of the bacteria or susceptibility to theantibiotic. A flat or substantially flat line followed by a delayedchange in ORP can indicate partial elimination of the bacteria (i.e.,time-shifted regrowth in the presence of the antibiotic) or intermediatesusceptibility to the antibiotic.

FIG. 9A illustrates a side view of one embodiment of a sensor 900. Thesensor 900 can be or refer to any of the sensors depicted in FIGS. 1,2A, 2B, 2C, 5, and 6 (including but not limited to sensor 116 of FIG. 1,2A, 2B, or 2C; the first sensor 508 or the second sensor 510 of FIG. 5or 6; and the third sensor 602, the fourth sensor 604, or the fifthsensor 606 of FIG. 6). The sensor 900 can be an electrochemical cellcomprising an active electrode 901 and an external reference electrode902. In some embodiments of the sensor 900, the active electrode 901 andthe external reference electrode 902 are the only electrodes of thesensor 900.

The active electrode 901 can extend from or be disposed on a substratelayer 904. The substrate layer 904 can be composed of, but is notlimited to, any non-conducting material such as a polymer, an oxide, aceramic, or a composite thereof. The electrochemical cell can besurrounded or contained by walls 906 configured to retain a sampledsolution 910. The walls 906 can be made of an inert or non-conductivematerial.

The sampled solution 910 can refer to any of the diluted sample 112, thetest solutions, the control solution 504, or an aliquot thereof. Atleast part of external reference electrode 902 can be in fluidcommunication or fluid contact with the sampled solution 910. Forexample, the external reference electrode 902 can extend into or beimmersed in the sampled solution 910. The external reference electrode902 can also have a stable or well-known internal voltage and the sensor900 can use the external reference electrode 902 to determine or measurea relative change in the potential of the active electrode 901. In oneembodiment, the external reference electrode 902 can be a standaloneprobe or electrode. In other embodiments, the external referenceelectrode 902 can be coupled to the parameter analyzer 118. In someembodiments, multiple sensors (including but not limited to any of thefirst sensor 508, the second sensor 510, the third sensor 602, thefourth sensor 604, or the fifth sensor 606) can share and use the sameexternal reference electrode 902.

In one embodiment, the external reference electrode 902 can be asilver/silver chloride (Ag/AgCl) electrode. In other embodiments, theexternal reference electrode 902 can comprise a saturated calomelreference electrode (SCE) or a copper-copper (II) sulfate electrode(CSE). The external reference electrode 902 can also be apseudo-reference electrode including any metal that is not part of theactive electrode such as platinum, silver, gold, or a combinationthereof; any metal oxide or semiconductor oxide material such asaluminum oxide, iridium oxide, silicon oxide; or any conductive polymerelectrodes such as polypyrrole, polyaniline, polyacetylene, or acombination thereof.

The active electrode 901 can comprise multiple conductive layers (e.g.,a stack of metallic layers) and a redox-active material 908 or layersuch as a gold layer, a platinum layer, a metal oxide layer, a carbonlayer, or a combination thereof on top of the multiple conductivelayers. In some embodiments, the metal oxide layer can comprise aniridium oxide layer, a ruthenium oxide layer, or a combination thereof.The parameter analyzer 118 can be coupled to the active electrode 901and the external reference electrode 902.

The parameter analyzer 118 can determine the ORP of the sampled solution910 by measuring the potential difference between the external referenceelectrode 902 and the active electrode 901 instantly or over a period oftime. As shown in FIG. 9A, the parameter analyzer 118 can be a voltmeteror any other type of high-impedance amplifier or sourcemeter. Thevoltmeter can measure a relative change in an equilibrium potential atan interface between the redox-active material 908 of the activeelectrode 901 and the sampled solution 910 containing electro-activeredox species. The solution characteristic of the sampled solution 910can change as the amount of electro-active redox species changes due tothe energy use, oxygen uptake or release, growth, or metabolism of theinfectious agents 102 in solution. For example, the amount ofelectro-active redox species in the sampled solution 910 can change as aresult of cellular activity undertaken by the infectious agents 102 insolution. As a more specific example, the amount of electron donors fromTable 1 (e.g., the amount of energy carriers such as nicotinamideadenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH₂)) inthe sampled solution 910 can change due to the growth or lack thereof ofthe infectious agents 102 in solution. Also, as another more specificexample, the amount of oxygen depleted in the sampled solution 910 canchange due to the growth or lack thereof of the infectious agents 102 insolution.

In one embodiment, the active electrode 901 can comprise a metalliclayer. The metallic layer can comprise a gold layer, a platinum layer,or a combination thereof. The active electrode 901 can also comprisemultiple layers comprising a semiconductor layer having a redox-activemetal oxide layer, such as iridium oxide or ruthenium oxide on top ofthe multiple layers. In other embodiments, the active electrode 901 cancomprise one or more metallic layers, one or more redox-active metaloxide layers, one or more semiconductor layers, or any combination orstacking arrangement thereof.

FIG. 9B illustrates a side view of another embodiment of the sensor 900having an on-chip reference electrode 912 disposed on the substratelayer 904 in lieu of the external reference electrode 902 of FIG. 9A. Insome embodiments of the sensor 900, the active electrode 901 and theon-chip reference electrode 912 are the only electrodes of the sensor900.

In these and other embodiments, the on-chip reference electrode 912 canbe coated by a polymeric coating. For example, the on-chip referenceelectrode 912 can be coated by a polyvinyl chloride (PVC) coating, aperfluorosulfonate coating (e.g., Nafion™), or a combination thereof.

The on-chip reference electrode 912 can serve the same purpose as theexternal reference electrode 902 except be fabricated on or integratedwith the substrate layer 904. The on-chip reference electrode 912 can belocated adjacent to or near the active sensor 120. The sensor 900 ofFIG. 9B can serve the same function as the sensor 900 of FIG. 9A.Similar to the active electrode 901 of FIG. 9B, the on-chip referenceelectrode 912 can also be in fluid communication or communication withthe sampled solution 910 retained within walls 906.

The on-chip reference electrode 912 can be comprised of a metal, asemiconductor material, or a combination thereof. The metal of theon-chip reference electrode 912 can be covered by an oxide layer, asilane layer, a polymer layer, or a combination thereof. In anotherembodiment, the on-chip reference electrode 912 can be a metal combinedwith a metal salt such as an Ag/AgCl on-chip reference electrode. Inanother embodiment, the on-chip reference electrode can be aminiaturized electrode with a well-defined potential. In someembodiments, multiple sensors can share and use the same on-chipreference electrode 912. The on-chip reference electrode 912 cancomprise a saturated calomel reference electrode (SCE) or acopper-copper (II) sulfate electrode (CSE). The on-chip referenceelectrode 912 can also comprise a pseudo-reference electrode includingany metal that is not part of the active electrode such as platinum,silver, gold, or a combination thereof; any metal oxide or semiconductoroxide material such as aluminum oxide, iridium oxide, silicon oxide; orany conductive polymer electrodes such as polypyrrole, polyaniline,polyacetylene, or a combination thereof.

Each of the individual variations or embodiments described andillustrated herein has discrete components and features which may bereadily separated from or combined with the features of any of the othervariations or embodiments. Modifications may be made to adapt aparticular situation, material, composition of matter, process, processact(s) or step(s) to the objective(s), spirit or scope of the presentinvention.

Methods recited herein may be carried out in any order of the recitedevents that is logically possible, as well as the recited order ofevents. For example, the flowcharts or process flows depicted in thefigures do not require the particular order shown to achieve the desiredresult. Moreover, additional steps or operations may be provided orsteps or operations may be eliminated to achieve the desired result.

It will be understood by one of ordinary skill in the art that all or aportion of the methods disclosed herein may be embodied in anon-transitory machine readable or accessible medium comprisinginstructions readable or executable by a processor or processing unit ofa computing device or other type of machine.

Furthermore, where a range of values is provided, every interveningvalue between the upper and lower limit of that range and any otherstated or intervening value in that stated range is encompassed withinthe invention. Also, any optional feature of the inventive variationsdescribed may be set forth and claimed independently, or in combinationwith any one or more of the features described herein.

All existing subject matter mentioned herein (e.g., publications,patents, patent applications and hardware) is incorporated by referenceherein in its entirety except insofar as the subject matter may conflictwith that of the present invention (in which case what is present hereinshall prevail). The referenced items are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such material by virtue of prior invention.

Reference to a singular item, includes the possibility that there areplural of the same items present. More specifically, as used herein andin the appended claims, the singular forms “a,” “an,” “said” and “the”include plural referents unless the context clearly dictates otherwise.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

This disclosure is not intended to be limited to the scope of theparticular forms set forth, but is intended to cover alternatives,modifications, and equivalents of the variations or embodimentsdescribed herein. Further, the scope of the disclosure fully encompassesother variations or embodiments that may become obvious to those skilledin the art in view of this disclosure. The scope of the presentinvention is limited only by the appended claims.

What is claimed is:
 1. A method of determining a concentration of aninfectious agent, the method comprising: diluting a sample comprisingthe infectious agent with a dilutive solution to yield a diluted sample;introducing the diluted sample to a sensor such that the diluted sampleis in fluid communication with a redox-active material of the sensor;and monitoring an oxidation reduction potential (ORP) of the dilutedsample over a period of time using at least one parameter analyzercoupled to the sensor to determine the concentration of the infectiousagent in the sample, wherein the ORP is monitored in the absence of anyadded reporter molecules in the diluted sample.
 2. The method of claim1, wherein the dilutive solution comprises growth media and the methodfurther comprises incubating the diluted sample at an elevatedtemperature.
 3. The method of claim 1, wherein the dilutive solutioncomprises at least one of deionized water and a saline solution and themethod further comprises: introducing the diluted sample to the sensorthrough a sample delivery conduit comprising growth media such that thediluted sample is mixed with the growth media; and incubating thediluted sample mixed with the growth media at an elevated temperature.4. The method of claim 3, wherein the growth media within the sampledelivery conduit is lyophilized or dried such that the growth mediamixes with the diluted sample.
 5. The method of claim 3, wherein thegrowth media within the sample delivery conduit is in aqueous form suchthat the growth media mixes with the diluted sample.
 6. The method ofclaim 1, further comprising determining the concentration of theinfectious agent using a standard curve generated by monitoring the ORPsof prepared cultures of the infectious agent in differentconcentrations.
 7. The method of claim 1, wherein the sample comprises abodily fluid, a wound swab or sample, a rectal swab or sample, anothertype of biological sample, a bacterial culture derived therefrom, or acombination thereof.
 8. The method of claim 7, wherein the bodily fluidcomprises urine, blood, sputum, saliva, breast milk, spinal fluid,semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleuralfluid, peritoneal fluid, pericardial fluid, amniotic fluid, cultures ofbodily fluid which has been tested positive for bacteria or bacterialgrowth, or a combination thereof.
 9. The method of claim 1, whereindiluting the sample with the dilutive solution further comprisesdiluting the sample to a dilution ratio between about 1:1 to about1:10000.
 10. The method of claim 1, wherein the sensor comprises anactive electrode and a reference electrode.
 11. The method of claim 1,wherein the infectious agent comprises bacteria.
 12. The method of claim1, wherein the infectious agent comprises fungus, mold, or a combinationthereof.
 13. The method of claim 1, wherein the redox-active materialcomprises a gold layer, a platinum layer, a metal oxide layer, a carbonlayer, or a combination thereof.
 14. A method of determining asusceptibility of an infectious agent to an anti-infective, the methodcomprising: diluting a sample comprising the infectious agent with adilutive solution to yield a diluted sample; separating the dilutedsample into a first aliquot and a second aliquot, wherein the secondaliquot is used as a control solution; mixing an anti-infective at afirst concentration into the first aliquot to yield a test solution;introducing the test solution to a first sensor such that the testsolution is in fluid communication with a redox-active material of thefirst sensor; introducing the control solution to a second sensor suchthat the control solution is in fluid communication with theredox-active material of the second sensor; monitoring an oxidationreduction potential (ORP) of the test solution and the control solutionover a period of time using one or more parameter analyzers coupled tothe first sensor, the second sensor, or a combination thereof, whereinthe ORPs are monitored in the absence of any added reporter molecules inthe test solution or the control solution; and comparing the ORP of thetest solution with the ORP of the control solution to determine thesusceptibility of the infectious agent to the anti-infective.
 15. Themethod of claim 14, further comprising: separating the diluted sampleinto a third aliquot; mixing the anti-infective at a secondconcentration into the third aliquot to yield another test solution;introducing the other test solution to a third sensor such that theother test solution is in fluid communication with the redox-activematerial of the third sensor; monitoring the ORP of the other testsolution over the period of time using the one or more parameteranalyzers coupled to the third sensor; and comparing the ORP of theother test solution with the ORPs of the test solution and the controlsolution to determine a degree of susceptibility of the infectious agentto the anti-infective, wherein the ORP is monitored in the absence ofany added reporter molecules in the other test solution.
 16. The methodof claim 14, wherein the dilutive solution comprises growth media andthe method further comprises incubating the test solution and thecontrol solution at an elevated temperature.
 17. The method of claim 14,wherein the dilutive solution comprises at least one of deionized waterand a saline solution; wherein mixing the anti-infective into the firstaliquot comprises delivering the first aliquot through a sample deliveryconduit comprising growth media and the anti-infective such that thegrowth media and the anti-infective are mixed into the first aliquot toyield the test solution; and further comprising introducing the secondaliquot through another sample delivery conduit comprising the growthmedia to yield the control solution; and incubating the test solutionand the control solution at an elevated temperature.
 18. The method ofclaim 17, wherein the growth media and the anti-infective within thesample delivery conduits are lyophilized or dried.
 19. The method ofclaim 17, wherein at least one of the growth media and theanti-infective within the sample delivery conduits are in aqueous form.20. The method of claim 14, wherein the sample comprises a bodily fluid,a wound swab or sample, a rectal swab or sample, another type ofbiological sample, a bacterial culture derived therefrom, or acombination thereof.
 21. The method of claim 20, wherein the bodilyfluid comprises urine, blood, sputum, saliva, breast milk, spinal fluid,semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleuralfluid, peritoneal fluid, pericardial fluid, amniotic fluid, cultures ofbodily fluid which has been tested positive for bacteria or bacterialgrowth, or a combination thereof.
 22. The method of claim 14, whereindiluting the sample with the dilutive solution further comprisesdiluting the sample to a dilution ratio between about 1:1 to about1:10000.
 23. The method of claim 14, wherein each of the first sensorand the second sensor comprises an active electrode and a referenceelectrode.
 24. The method of claim 14, wherein the redox-active materialcomprises a gold layer, a platinum layer, a metal oxide layer, a carbonlayer, or a combination thereof.
 25. The method of claim 14, wherein theinfectious agent comprises bacteria.
 26. The method of claim 25, whereinthe anti-infective comprises a bacteriostatic anti-infective, abactericidal anti-infective, or a combination thereof.
 27. The method ofclaim 14, wherein the infectious agent comprises fungi.
 28. The methodof claim 27, wherein the anti-infective comprises an anti-fungal.